DNA Cleavage and Cytotoxic Activity of Copper(II) Complexes Based on Reduced Schiff Bases Derived From Salicylaldehyde and Amino Acids

Synthesis of ligands was performed in two steps. In the first step, Schiff bases were prepared from salicylaldehyde and sodium salts of linear amino acids (β-alanine, γ-aminobutanoic acid, 5-aminopentanoic acid, and 6-aminohexanoic acid) by condensation reaction. In the second step, a reduction of the imine double bond with NaBH₄ was performed to obtain reduced Schiff base ligands. The copper(II) complexes were prepared from reduced Schiff bases and copper(II) acetate in methanol as green powders. The prepared ligands were characterized by nuclear magnetic resonance (NMR), IR, and elemental analysis, and the complexes were characterized by IR and elemental analysis.

Scheme of synthesis of copper(II) complexes and the listed prepared complexes:

The complexes (LL4), (LL5), and their free ligands were prepared by Dr. Lucia Lintnerová and have been published in a previous study. (5)

Two hundred and fifty nanograms of DNA plasmid pBBR322 (in 25 mM Tris HCl buffer solution, pH 7.5) was treated with copper(II) complexes (suspended in DMSO) for 5 h at 37 °C. Each reaction was then quenched by adding 4 μL of a Smart Glow Loading Dye^® (0.01% Safe Green dye, 50% glycerol, and 250 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0), and the digested samples were electrophoresed on 0.9% agarose gel containing TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA) at 55 V/40 mA for about 1.5 h. TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA) was used as the “running buffer.” The resulting cleaved fragments which correspond to the cleaved form II. (open circular form) and cleaved form III. (linear form) were visualized by ultraviolet (UV) light (6,7). Quantitative evaluation of cleavage activity was performed using the “gelQUANT software” (Cleaver Scientific, UK).

Saccharomyces cerevisiae (3,8) cells (CCY 21-4-64; Libáň, Czech Republic) were cultured on Sabouraud dextrose agar with chloramphenicol (Merck, USA) and liquid YPD medium (Sigma-Aldrich, USA) at 28°C/24 h. In the exponential growth phase, the cells were centrifuged (8,000 g/3 min), washed twice with physiologic solution (FS 0.9% NaCl), and pipetted at a concentration of 1 × 10⁹ cells/ml into sterile microtubes. Sterile FS and copper(II) complexes (1 × 10⁻³ M, 3 × 10⁻³ M, and 5 × 10⁻³ M) were added to the centrifuged biomass and cultured at 28°C/3 h. After 3 h, 0.1 ml of resazurin (Sigma, USA) was added to the samples and they were incubated for 2 h. After 2 h, the biomass was separated from the supernatant by centrifugation (8,000 g/5 min). The separated solutions were diluted in a ratio of 1:1 with FS and measured (600 nm/resazurin, 570 nm/resorufin = metabolized resazurin) on UVVIS spectrophotometer (Thermo Scientific, USA) using a 1-cm-long cuvette (8,9). Control samples used: C– negative control (1 ml 0.9% NaCl + 0.1 ml resazurin); C+ positive control (S. cerevisiae cells 1 × 10⁹/ml + 1 ml 0.9% NaCl + 0.1 ml resazurin) measured at t = 3 h.

Cells’ innate metabolism was measured by using an established formula for reduction percentage. Reduction percentage was calculated by the following formula: Red[%]=εOX_600 nm×A570 nm_sample-εOX_570 nm×A600 nm_sampleεRED_570 nm×A600 nm_C− − εRED_600 nm×A570 nm_C−×100 {\rm{Red}}[\% ] = {{{\varepsilon _{{\rm{OX}}\_600\,{\rm{nm}}}} \times {{\rm{A}}_{570{\rm{nm}}\_{\rm{sample -}}}}{\varepsilon _{{\rm{OX}}\_570\,{\rm{nm}}}} \times {{\rm{A}}_{600{\rm{nm}}\_{\rm{sample}}}}} \over {{\varepsilon _{{\rm{RED}}\_570\,{\rm{nm}}}} \times {{\rm{A}}_{600{\rm{nm}}\_{\rm{C}} - \,\,\,\,\,\, -}}\,\,{\varepsilon _{{\rm{RED}}\_600\,{\rm{nm}}}} \times {{\rm{A}}_{570{\rm{nm}}\_{\rm{C}} -}}}} \times 100

The cytotoxic activity of copper(II) complexes was calculated according to the formula Cy [%]=100−Red [%] {\rm{Cy}}\,[\% ] = 100 - {\mathop{\rm Red}\nolimits} \,[\% ]

Molar extinction coefficients for reduced and oxidized resazurin:

Nuclease activity was recorded for all tested complexes, which increased predominantly with increasing concentration of the copper complex. In complexes LL2, LL3, LL4, and LL5, plasmid DNA (pDNA) was split into an open circular form (form II.) in the amount of LL2: 35.7%–73.8% (1 × 10⁻³–5 × 10⁻³ M); LL3: 7.8%–15.2% (1 × 10⁻³–5 × 10⁻³ M); LL4: 37.1%–47.9% (1 × 10⁻³–5 × 10⁻³ M); LL5 21.3% (1 × 10⁻³ M). Complex LL5 had the best cleavage activity (compared to negative control), which, in the concentration of 3 × 10⁻³ and 5 × 10⁻³ M, completely cleaved pDNA into a linear form (100% form III.) from the original double-stranded supercoiled form (form I.) of pDNA (Figs 1–4).

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

The cytotoxic activity of the individual complexes is shown in Fig. 5. All tested copper complexes showed a high cytotoxic activity above the level of 90% compared to the positive control (8%). Cytotoxicity test is based on the ability of living cells (redox reaction in mitochondria) to reduce blue resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) into pink resorufin (7-hydroxy-3H-phenoxazin-3-one) (8,10). Bioreduction of the dye by viable cells reduces the amount of the oxidized form (blue) and concomitantly increases the amount of its fluorescent intermediate (pink), indicating the degree of cytotoxicity caused by the test material (2,11). Cytotoxic activity was recorded for all tested copper(II) complexes in concentrations of 1 × 10⁻³, 3 × 10⁻³, and 5 × 10⁻³ after only 3 h of incubation with S. cerevisiae cells.

Figure 5.