DNA binding properties of ruthenium arene complex, genotoxicity and in vitro cytotoxicity

2, 4-Diamino-1,3,5-triazine derivatives have been shown to display antitumour and biological activity. In this field, there is a growing interest on the synthesis of novel diaminotriazine derivatives such as the 2-pydaT complex ligand used in this work. On the other hand, ruthenium complexes have attracted much attention recently due to their antitumour potential. In this context, arene Ru (II) complexes bearing a diamino- triazine derivative have been synthesized and characterized by Prof. Espino´s group in Burgos University. One of them was used for DNA binding and genotoxicity studies. To a first place, a stability study of the complex has been carried out. This compound undergoes hydrolyses both in water and in basic media to yield Ru-H2O and Ru-OH, respectively. The two processes were detected by NMR and UV-Vis measurements. In the presence of NaCl and pH = 7.0, the Ru-Cl complex is the only species present. To a second place, the interaction of Ru-Cl with CT-DNA was studied by a number of available techniques. The binding constants evaluated at different ionic strengths by spectrofluorometric titrations evince a notable electrostatic contribution. Spectrophotometric titrations and melting assays were also carried out. Ru-Cl showed no contribution to the thermal stabilization of the double helix. The conformational transitions of the interaction were studied by circular dichroism and viscosity experiments. The data gathered suggest that at least two different complexes are formed, depending on both the Ru-Cl and DNA concentrations ratio. The observation that the relative viscosity slightly decreased with increasing Ru-Cl amounts, rules out intercalation as the mode of binding. These findings support the assumption that groove binding and external electrostatic binding take place as a function of the Ru-Cl / DNA ratio. Additionally, the genotoxic activity of the Ru-Cl complex was tested in V79 cell line using the cytokinesis-block micronucleus assay, a widely applied technique for screening of chemicals. Surprisingly, the results achieved for all the doses tested suggest that Ru-Cl is by no means genotoxic under the conditions used. The in vitro cytotoxicity of the arene ruthenium complexes as well as the diamino-triazine ligands alone, characterized in V-79 cell line by the MTT cell proliferation assay with 24 hours exposure time, have discarded any cytotoxic activity. In line with the features described of ruthenium arene and diamoni-triazine derivatives, it was thought reasonable to assume that these compounds could display cytotoxic and genotoxic activity. Also, Ru-Cl can interact with DNA outside the cell. The existence of another brown species, detected in basic media by NMR, can shed light into the absence of correlation of the DNA binding studies with the biological assays, in which it was observed that the cell media turned to brown. These findings suggest that the brown species is formed into the cell as a consequence of the Ru-Cl complex metabolization.