In situ electrochemical evaluation of anticancer drug temozolomide and its metabolites-DNA interaction.

Temozolomide (TMZ) is an antineoplastic alkylating agent with activity against serious and aggressive types of brain tumours. It has been postulated that TMZ exerts its antitumor activity via its spontaneous degradation at physiological pH. The in vitro evaluation of the interaction of TMZ and its final metabolites, 5-aminoimidazole-4-carboxamide (AIC) and methyldiazonium ion, with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues. The results obtained revealed the decrease of the dsDNA oxidation peaks with incubation time, showing that TMZ and AIC/methyldiazonium ion interact with dsDNA causing its condensation. Furthermore, the experiments of the in situ TMZ and AIC/methyldiazonium ion-dsDNA interaction using the multilayer dsDNA-electrochemical biosensor confirmed the condensation of dsDNA caused by these species and showed evidence for a specific interaction between the guanosine residues and TMZ metabolites, since free guanine oxidation peak was detected. The oxidative damage caused to DNA bases by TMZ metabolites was also detected electrochemically by monitoring the appearance of the 8-oxoguanine/2,8-dyhydroxyadenine oxidation peaks. Nondenaturing agarose gel electrophoresis of AIC/methyldiazonium ion-dsDNA samples confirmed the occurrence of dsDNA condensation and oxidative damage observed in the electrochemical results. The importance of the dsDNA-electrochemical biosensor in the in situ evaluation of TMZ-dsDNA interactions is clearly demonstrated.

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals on a boron-doped diamond electrode.

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals has been directly demonstrated on a boron-doped diamond electrode. The DNA-electrochemical biosensor incorporates immobilized double-stranded DNA (dsDNA) as molecular recognition element on the electrode surface, and measures in situ specific binding processes with dsDNA, as it is a complementary tool for the study of bimolecular interaction mechanisms of compounds binding to DNA and enabling the screening and evaluation of the effect caused to DNA by radicals and health hazardous compounds. Oxidants, particularly reactive oxygen species (ROS), play an important role in dsDNA oxidative damage which is strongly related to mutagenesis, carcinogenesis, autoimmune inflammatory, and neurodegenerative diseases. The hydroxyl radical is considered the main contributing ROS to endogenous oxidation of cellular dsDNA causing double-stranded and single-stranded breaks, free bases, and 8-oxoguanine occurrence. The dsDNA-electrochemical biosensor was used to study the interaction between dsDNA immobilized on a boron-doped diamond electrode surface and in situ electrochemically generate hydroxyl radicals. Non-denaturing agarose gel-electrophoresis of the dsDNA films on the electrode surface after interaction with the electrochemically generated hydroxyl radicals clearly showed the occurrence of in situ dsDNA oxidative damage. The importance of the dsDNA-electrochemical biosensor in the evaluation of the dsDNA-hydroxyl radical interactions is clearly demonstrated.

In situ evaluation of chromium-DNA damage using a DNA-electrochemical biosensor.

The in situ evaluation of the direct interaction of chromium species with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine bases. The results obtained revealed the interaction with dsDNA of the Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation by O(2) dissolved in the solution bound to dsDNA. This interaction leads to different modifications and causes oxidative damage in the B-DNA structure. Using polyhomonucleotides of guanine and adenine, it was shown that the interaction between reactive intermediates Cr(IV) and Cr(V)-DNA causes oxidative damage and preferentially takes place at guanine-rich segments, leading to the formation of 8-oxoguanine, the oxidation product of guanine residues and a biomarker of DNA oxidative damage. The interaction of Cr(VI) with dsDNA causes breaking of hydrogen bonds, conformational changes, and unfolding of the double helix, which enables easier access of other oxidative agents to interact with DNA, and the occurrence of oxidative damage to DNA.

DNA-electrochemical biosensors: AFM surface characterisation and application to detection of in situ oxidative damage to DNA.

In recent years increased attention has been focused on the ways in which drugs interact with DNA, with the goal of understanding the toxic as well as chemotherapeutic effects of many molecules. The development of fast and accurate methods of DNA damage detection is important, especially caused by anticancer drugs or hazard compounds. The DNA-electrochemical biosensor is a very good model for evaluation of nucleic acid damage, and electrochemical detection a particularly sensitive and selective method for the investigation of specific interactions. The electrochemical sensor for detecting DNA damage consists of a glassy carbon electrode with DNA immobilized on its surface. The starting materials or the redox reaction products can be pre-concentrated on the dsDNA-biosensor surface, enabling electrochemical probing of the presence of short-lived radical intermediates and of their damage to dsDNA. AFM images were used to characterize different procedures for immobilization of nanoscale DNA surface films on carbon electrodes before and after interaction with hazard compounds. The electrochemical transduction is dynamic in that the electrode is itself a tuneable charged reagent as well as a detector of all surface phenomena, which greatly enlarges the electrochemical biosensing capabilities.