Study of interaction of antimutagenic 1,4-dihydropyridine AV-153-Na with DNA-damaging molecules and its impact on DNA repair activity

Chemicals

AV-153-Na and AV-154-Na were synthesized in the Laboratory of Membrane Active Compounds at the Latvian Institute of Organic Synthesis. Structures of the AV-153-Na and AV-154-Na are given in Fig. 1; the synthesis of the compounds was performed essentially as described (Dubur & Uldrikis, 1969). Tris base, ferrous sulphate, 5,5-dimethylpyrroline-N-oxide (DMPO), Triton-X-100, human serum albumin (HSA), Na₂EDTA, LiCl, NaCl, CaCl₂ and other inorganic salts were purchased from Sigma-Aldrich (Taufkirchen, Germany). Peroxynitrite was synthesized as described by Robinson & Beckman (2005).

Cell culture

HeLa cells (Biomedical Research and Study Centre, Riga, Latvia) were grown in DMEM + GlutamaxTM–I, F-12 Nut-Mix (1×) (Sigma-Aldrich, Taufkirchen, Germany) + 10% fetal bovine serum (Sigma-Aldrich, USA), at 37 °C in a humidified atmosphere containing 5% CO₂.

Fenton reaction—ESR measurements

For the Fenton reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + ⋅OH + OH⁻), 80 µl of reaction mixture containing 250 µM ferrous sulphate, 250 µM H₂O₂, 80 mM spin trap DMPO, and 1 mM of 1,4 DHP was transferred to a micro pipettes tube for measurement of the electron spin resonance (ESR) spectra of DMPO-OH radicals. ESR spectra of the spin trap and radical complex were recorded at room temperature using an EMX-plus EPR spectrometer (Bruker, Germany). The EPR instrumental settings for field scan were as follows: field sweep—100G; microwave frequency—9.84 GHz; microwave power—15.9 mW; modulation amplitude—1 G; conversion time—163 ms; time constant—327 ms; sweep time –83 s; receiver gain–1⋅10⁴; resolution—512 points for 1 scan.

The single cell electrophoresis (comet assay)

The single cell electrophoresis (comet assay) was performed as described previously (Leonova et al., 2016)

UV/VIS spectroscopic measurement of peroxynitrite decomposition

The rate of peroxynitrite (0.38 mM) decomposition in the presence or in the absence of the 1,4-DHP (0.16 mM) was followed at 302 nm (absorbance peak for the peroxynitrite anionic form) in 10 mM Tris pH 10 buffer on Perkin Elmer Lambda 25 UV/VIS spectrophotometer (Carballal, Bartesaghi & Radi, 2014). The average rate of reactions were calculated according to the formula V =  ± ((C₂ − C₁)∕(t₂ − t₁)) =  ± (ΔC∕Δt), where C₁ was the concentration of peroxynitrite in the beginning of reaction, and C₂ the concentration of peroxynitrite at the end of the reaction; Δt: 20 min.

Circular dichroism spectroscopy

CD spectra were recorded on a Chirascan CS/3D spectrometer (Applied Photophysics, Surrey, UK); CD spectra of HSA in the absence or in the presence of AV-153-Na salts were recorded in PBS buffer, pH 7.4 in a range of 200–260 nm. A 300 nM HSA solution was titrated with AV-153-Na (1 µM at each step) in a quartz cell of 10 mm path-length at room temperature. The parameters for the spectra were as follows: scan rate—200 nm min⁻¹, averaging time— 0.125 s, bandwidth—1 nm; one recorded spectrum is the average of four scans.

Fluorescence spectroscopic measurements

Spectrofluorimetric analyses were performed on a Fluoromax-3 (Horiba JOBIN YVON, Shanghai, China), essentially as described (Osina et al., 2017).

Cell treatment and nuclear extract preparation for repair reactions

HeLa cells were incubated with 50 nM of AV-153-Na for 3, 12 or 24 h, washed with PBS, trypsinized, suspended in PBS and pelleted by low-speed centrifugation. Cell pellets were incubated in ice for 20 min in 1.25 mL of ice-cold buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.01% Triton X-100, 0.5 mM DTT, 0.5 mM PMSF) and vortexed for 30 s. After centrifugation for 5 min at 5,000 rpm at 4 °C, the nuclei were suspended in 31.25 µL of ice-cold buffer B (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 400 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, antiproteases (Complete-mini, Roche, Meylan, France) and 0.5 mM PMSF). The nuclear membrane lysis was completed by incubation for 20 min on ice, followed by two cycles of freezing-thawing at −80 °C and 4 °C respectively. Debris was eliminated by centrifugation for 10 min at 13,000 rpm at 4 °C. The supernatant was stored frozen in 10 µl aliquots at −80 °C. Protein content was determined using the BCA kit (Interchim, Montluçon, France). Typical protein content was 0.8 mg/mL.

Assays of the activity of DNA repair enzymes

The impact of the tested compounds on activity of glycosylases/AP endonucleases belonging to Base Excision Repair and on Excision/Synthesis Repair activities was performed using Glyco-SPOT (Pons et al., 2010) and ExSy-SPOT assays (Forestier et al., 2012), respectively (LXRepair, Grenoble, France). These assays allowed quantifying different DNA repair activities from extracts prepared from treated and non-treated cells.

The former chip, which is a multiplex, on-support, oligonucleotide (ODN) cleavage assay, reveals excision activities against 8-oxoguanine paired with C (8-oxoG-C), A paired with 8oxoguanine (A-8oxoG), ethenoadenine (EthA-T), thymine glycol (Tg-A), uracil (paired either with G or A (U-G and U-A, respectively)), hypoxanthine (Hx-T), and abasic sites (THF-A). Cleavage of the lesions by the enzymes contained in the extracts released the fluorescence attached to the lesion containing ODNs.

Repair reactions were conducted for 1 h at 37 °C with 15 µg/mL of protein in 80 µL of excision buffer (10 mM Hepes/KOH pH 7.8, 80 mM KCl, 1 mM EGTA, 0.1 mM ZnCl₂, 1 mM DTT, 0.5 mg/mL BSA). After 3 washes, 5 min at room temperature in PBS containing 0.2 M NaCl and 0.1% Tween 20, the spots fluorescence was quantified using the Innoscan scanner from Innopsys (Toulouse, France). Each extract was run in duplicate. The results between the replicates (4 spot fluorescence) were normalized using the NormalizeIt software as described by Millau et al. (2008).

Wells incubated with the excision buffer served as reference (100% fluorescence) to calculate the lesions percentage of cleavage in the wells incubated with the extracts. Non-specific cleavage of the control ODN (Lesion_Free ODN) was also taken into account to calculate the percentage of excision of each lesion using the following formula: (100 ×(1−percentage of fluorescence of Lesion_ODN/percentage of fluorescence of Lesion_Free ODN)).

The ExSy-SPOT assay quantified Excision/Synthesis Repair of 8-oxoguanine (8oxoG), alkylated bases (AlkB) and abasic sites (AbaS), incorporated into different supercoiled plasmid DNA. The principle of the methods is described by Millau et al. (2008). Extracts (0.1 mg/mL) incubated on the biochip where the different plasmid preparations were immobilized at specific sites for 3 h at 30 °C in reaction buffer (40 mM Hepes KOH pH 7.8, 7 mM MgCl2, 0.5 mM DTT, 0.25 µM dATP, 0.25 µM dTTP, 0.25 µM dGTP, 3.4% glycerol, 12.5 mM phosphocreatine (Sigma, Taufkirche, Germany), 2 mM EDTA, 50 µg/mL creatine phosphokinase, 0.1 mg/mL BSA) containing 1 mM ATP (Amersham, England) and 1.25 µM dCTP-Cy3. After washing for 3 × 5 min in H₂O (MilliQ), the total fluorescence intensity of each spot was quantified using the Innoscan scanner from Innopsys (Toulouse, France). Each extract was run in duplicate and data were normalized using the NormalizeIt software as described by Millau et al. (2008). Results were expressed as Fluorescence Intensity (FI).

Laser confocal scanning microscopy

For imaging, the HeLa cells were seeded in 4 well Nunc Lab-Tek Permanox Chamber slide (Thermo Scientific Nunc, Pittsburg, PA, USA) and cultivated for 24 h as described above. Subsequently, the cells were washed twice with PBS for 5 min and then incubated with 1 mM AV-153-Na in PBS for 16 h in a CO₂ incubator at 37 °C and 5% CO₂. After incubation, the cells were washed with PBS for 5 min and fixed in 70% ethanol for 0.5 h at room temperature. Slides were rinsed with PBS for 5 min and counterstain chromatin was dyed with 15 µM propidium iodide (PI) in PBS for 0.5 h, then washed twice with PBS for 5 min. Slides were analyzed using a Leica DM RA-2 microscope equipment with a TCS-SL confocal scanning head (Leica Microsystems, Bannockburn, IL, USA). Images were collected with a Leica 40× HCX PL Fluator objective (NA = 0.75) and 100× HCX PIAPO oil immersion objective (NA = 1.40). AV-153-Na and propidium iodide were excited with a 488 nm band from a four-line argon ion laser. AV-153-Na fluorescence was detected between 510 and 560 nm, propidium iodide fluorescence was detected between 600 and 650 nm. Cell shapes were controlled with reflected light 475–505 nm. Cells were scanned along the Z-axis with a step size—0.5 µm.

Statistical analysis

The values of DNA damage assayed by single gel electrophoresis are represented as the mean ± standard error of the mean (SEM). The data were subjected to the one-way analysis of variance (ANOVA), followed by the Tukey multiple comparisons test and the data were considered as significant at p < 0.05.

Study of chemical interactions of AV-153-Na with DNA-damaging agents

Taking into account capability of the AV-153-Na to decrease DNA damage produced by hydrogen peroxide (Ryabokon et al., 2008; Ryabokon et al., 2005) we have tested the ability of the AV-153-Na to scavenge free radicals. The OH radical produced in Fenton reaction, was tested by the ESR method. We have tested AV-153-Na at 1,000 µM concentration. The signals of the second component of the EPR spectra were measured on the 3rd min (I₃) and the 5th min (I₅) and the difference between I₃ and I₅ was calculated (Fig. 1). Scavengers of OH radicals should increase the difference between I₃ and I₅. Representative kinetics of the decrease of EPR signal intensity is shown in Fig. 1C. The impact of AV-153-Na on the signal intensity is minimal, AV-154-Na does not produce any effect at all. Thus these 1,4-DHP derivatives are very weak radical scavengers.

Published data indicate the ability of some 1,4-DHP to scavenge peroxynitrite chemically (Lopez-Alarcon et al., 2004). We also tested the ability of the AV-153-Na salts to degrade peroxynitrite chemically by studying the kinetics of decomposition of peroxynitrite in the presence of DHP followed by means of spectrophotometry. The curves are presented in Fig. 2. The average rate of spontaneous decomposition of peroxynitrite at concentration 0.38 mM was 0.0157 µmol/µl min. AV-153-Na did not affect the time of decomposition of peroxynitrite; it remained 0.0157 µmol/µl min.

Intracellular localization of AV-153-Na and protein binding

As the capability of AV-153-Na to bind DNA in vivo was proven previously (Buraka et al., 2014), its antimutagenic effect could not be attributed to chemical interactions with DNA-damaging agents (see above) we needed evidence whether the compound could reach cell nucleus in the cells where it could interact with DNA. We have tried to answer this question by profiting of the intrinsic fluorescence of the compound with the aid of laser confocal scanning microscopy. Results are presented in Figs. 3A and 3C. In most cells AV-153-Na heavily stains the cytoplasm; however, some fluorescence is visible also in the nucleus, mainly in the nucleolus. Staining of cytoplasmic structures raised the question about capability of the compound to bind proteins. Fluorescence titration of AV-153-Na with human serum albumin gave a positive answer to the question - fluorescence of the compound increased in the presence of HSA. Results of CD confirmed the ability to bind a protein (Figs. 3D and 3E). The addition of the compound to the HSA solution decreased CD signals in both minimal bands (208 and 222 nm) in the presence of AV-153-Na, which suggests binding with HSA, causing protein conformational changes due to a slight protein unfolding (Wang et al., 2008).

Impact of AV-153-Na on DNA damage and DNA repair systems

The DNA-protecting action of AV-153 salts against peroxynitrite-induced damage was tested in living cells. Results of the comet assay experiments performed on HeLa cell treated with peroxynitrite alone or in the presence of AV-153-Na are presented in Fig. 4. Treatment with peroxynitrite drastically increased the levels of DNA damage. AV-153-Na reduced the extent of DNA damage produced by peroxynitrite. Pre-incubation with the compound at concentrations 50 nM for 45 min appeared to produce significant effects (Fig. 4A). When administered simultaneously with peroxynitrite, the compound produced a much weaker protective effect. AV-154-Na, which does not bind DNA (E Leonova et al., 2018, unpublished data), did not protect the cells against peroxynitrite (Fig. 4C).

Effects of AV-153-Na on the activity of DNA repair enzymes were tested using Glyco-SPOT and ExSy-SPOT assays. Longer pre-incubation times were chosen to reveal possible changes in protein expression.

The Glyco-SPOT assay revealed a specific and significant decrease of Tg (thymine glycol) repair by AV-153-Na, which manifested itself when an extract with a higher concentration of protein (15 µg/ml) was used in the assay, and a trend for inhibition of enzymes involved in U-G and U-A repair (Fig. 5). Other glycosylases/AP endonucleases activities were not affected.

Results with ExSy-SPOT assay appear to be more interesting in this sense. AV-153-Na stimulates the excision/synthesis repair of lesions repaired by Base Excision Repair (8-oxoG, abasic sites and alkylated bases (Fig. 6)). As this stimulating effect is not detected with the Glyco-SPOT assay, it involves either the synthesis step of the repair process or alternative repair pathways able to handle oxidative lesions.