Electrochemistry of chemotherapeutic alkylating agents and their interaction with DNA.

Alkylating agents were among the first anticancer drugs to be discovered and continue to be the most commonly used in chemotherapy. They are electrophiles that react with the ring nitrogen and extracyclic oxygen atoms of DNA bases, forming covalent adducts that further lead to cross-linking of DNA strands, abnormal base pairing or DNA strand breaks. The investigation and quantitative analysis of alkylating agents in biological samples are essential for monitoring the therapy progression and efficiency, understanding their pharmacokinetics and develop new more effective and specific chemotherapeutical drugs. Among biotechnological methods, electrochemical techniques are particularly important in pharmaceutical medicine, owing to their rapid detection, great sensitivity, robustness, exceptional detection limits, ability to be used with small analyte volumes in turbid biofluids, and easy adaptability to miniaturization and point-of-care (POC) testing. This article provides first an exhaustive review concerning the electrochemical methods of characterization and quantification of different classes of chemotherapeutic alkylating agents (triazenes and hydrazines, nitrosoureas, nitrogen mustards, oxazaphosphorines, alkyl alkane sulfonates and ethylene imines) in standard samples, pharmaceutical formulations and biological matrixes. The second part of the article focuses on the recent electrochemical methodologies and DNA-electrochemical biosensors developed to study the interaction of alkylating agents with DNA. These studies are relevant for obtaining real-time details about the alkylating agents' mechanism of action and for assessing the oxidative DNA damage they cause, important for the development of improved antineoplastic drugs.

DNA Electrochemical Biosensors for In Situ Probing of Pharmaceutical Drug Oxidative DNA Damage.

Deoxyribonucleic acid (DNA) electrochemical biosensors are devices that incorporate immobilized DNA as a molecular recognition element on the electrode surface, and enable probing in situ the oxidative DNA damage. A wide range of DNA electrochemical biosensor analytical and biotechnological applications in pharmacology are foreseen, due to their ability to determine in situ and in real-time the DNA interaction mechanisms with pharmaceutical drugs, as well as with their degradation products, redox reaction products, and metabolites, and due to their capacity to achieve quantitative electroanalytical evaluation of the drugs, with high sensitivity, short time of analysis, and low cost. This review presents the design and applications of label-free DNA electrochemical biosensors that use DNA direct electrochemical oxidation to detect oxidative DNA damage. The DNA electrochemical biosensor development, from the viewpoint of electrochemical and atomic force microscopy (AFM) characterization, and the bottom-up immobilization of DNA nanostructures at the electrode surface, are described. Applications of DNA electrochemical biosensors that enable the label-free detection of DNA interactions with pharmaceutical compounds, such as acridine derivatives, alkaloids, alkylating agents, alkylphosphocholines, antibiotics, antimetabolites, kinase inhibitors, immunomodulatory agents, metal complexes, nucleoside analogs, and phenolic compounds, which can be used in drug analysis and drug discovery, and may lead to future screening systems, are reviewed.

Nanostructured material-based electrochemical sensing of oxidative DNA damage biomarkers 8-oxoguanine and 8-oxodeoxyguanosine: a comprehensive review.

Oxidative DNA damage plays an important role in the pathogenesis of various diseases. Among oxidative DNA lesions, 8-oxoguanine (8-oxoG) and its corresponding nucleotide 8-oxo-2'-deoxyguanosine (8-oxodG), the guanine and deoxyguanosine oxidation products, have gained much attention, being considered biomarkers for oxidative DNA damage. Both 8-oxoG and 8-oxodG are used to predict overall body oxidative stress levels, to estimate the risk, to detect, and to make prognosis related to treatment of cancer, degenerative, and other age-related diseases. The need for rapid, easy, and low-cost detection and quantification of 8-oxoG and 8-oxodG biomarkers of oxidative DNA damage in complex samples, urine, blood, and tissue, caused an increasing interest on electrochemical sensors based on modified electrodes, due to their high sensitivity and selectivity, low-cost, and easy miniaturization and automation. This review aims to provide a comprehensive and exhaustive overview of the fundamental principles concerning the electrochemical determination of the biomarkers 8-oxoG and 8-oxodG using nanostructured materials (NsM), such as carbon nanotubes, carbon nanofibers, graphene-related materials, gold nanomaterials, metal nanoparticles, polymers, nanocomposites, dendrimers, antibodies and aptamers, and modified electrochemical sensors.

Natural phenolic antioxidants electrochemistry: Towards a new food science methodology.

Natural phenolic compounds are abundant in the vegetable kingdom, occurring mainly as secondary metabolites in a wide variety of chemical structures. Around 10,000 different plant phenolic derivatives have been isolated and identified. This review provides an exhaustive overview concerning the electron transfer reactions in natural polyphenols, from the point of view of their in vitro antioxidant and/or pro-oxidant mode of action, as well as their identification in highly complex matrixes, for example, fruits, vegetables, wine, food supplements, relevant for food quality control, nutrition, and health research. The accurate assessment of polyphenols' redox behavior is essential, and the application of the electrochemical methods in routine quality control of natural products and foods, where the polyphenols antioxidant activity needs to be quantified in vitro, is of the utmost importance. The phenol moiety oxidation pathways and the effect of substituents and experimental conditions on their electrochemical behavior will be reviewed. The fundamental principles concerning the redox behavior of natural polyphenols, specifically flavonoids and other benzopyran derivatives, phenolic acids and ester derivatives, quinones, lignins, tannins, lignans, essential oils, stilbenes, curcuminoids, and chalcones, will be described. The final sections will focus on the electroanalysis of phenolic antioxidants in natural products and the electroanalytical evaluation of in vitro total antioxidant capacity.

Calcium channel blocker lercanidipine electrochemistry using a carbon black-modified glassy carbon electrode.

Lercanidipine, a third-generation dihydropyridine calcium L-type channel blocker, redox behavior at different carbon electrode materials, in a wide pH range, using cyclic, square-wave, and differential pulse voltammetry, was studied. A comparison was made between unmodified glassy carbon electrode (GCE) and boron-doped diamond electrode (BDDE), and GCE and BDDE modified with a carbon black (CB) nanoparticle embedded within a dihexadecylphosphate (DHP) nanostructured film (CB-DHP/GCE and CB-DHP/BDDE). Lercanidipine oxidation, for 3.4 < pH < 9.5, is an irreversible, diffusion-controlled, pH-dependent process that occurs in two consecutive steps, with the transfer of one electron and one proton, at the N1 and C4 positions in the 1,4-dihydropyridine ring. For pH > 9.5, both oxidation processes are pH-independent and a pKa = 9.40 was determined. Lercanidipine reduction at pH = 7.0 is an irreversible process, and the lercanidipine reduction products are electroactive and follow a reversible electron transfer reaction. Lercanidipine electroanalytical determination, at a nanostructured GCE modified with a CB-DHP film (CB-DHP/GCE), with no need for N2 purging, with a detection limit of 0.058 µM (3.58 × 10-5 g L-1) and a quantification limit of 0.176 µM (1.08 × 10-4 g L-1), was achieved. Graphical abstract.

Caveolin proteins electrochemical oxidation and interaction with cholesterol.

Caveolae consist in lipid raft domains composed of caveolin proteins, cholesterol, glycosphingolipids, and GPI-anchored proteins. Caveolin proteins present three different types, caveolin 1 (CAV-1), caveolin 2 (CAV-2) and caveolin 3 (CAV-3), with a very similar structure and amino acid composition. The native caveolin proteins oxidation mechanism was investigated for the first time, at a glassy carbon electrode, using cyclic, square wave and differential pulse voltammetry. The three native caveolin proteins oxidation mechanism presented only one tyrosine and tryptophan amino acid residues oxidation peak. Denatured caveolin proteins presented also the tyrosine, tryptophan and cysteine amino acid residues oxidation peaks. The reverse cholesterol transport is related to caveolae and caveolin proteins, and CAV-1 is directly connected to cholesterol transport. The influence of cholesterol on the three caveolin proteins electrochemical behaviour was evaluated. In the absence and in the presence of cholesterol, significant differences in the CAV-1 oxidation peak current were observed.

Electrochemical and AFM Characterization of G-Quadruplex Electrochemical Biosensors and Applications.

Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited.

Electrochemistry of Alzheimer Disease Amyloid Beta Peptides.

Alzheimer's disease (AD) is a widespread form of dementia that is estimated to affect 44.4 million people worldwide. AD pathology is closely related to the accumulation of amyloid beta (Aß) peptides in fibrils and plagues, the small oligomeric intermediate species formed during the Aß peptides aggregation presenting the highest neurotoxicity. This review discusses the recent advances on the Aß peptides electrochemical characterization. The Aß peptides oxidation at a glassy carbon electrode occurs in one or two steps, depending on the amino acid sequence, length and content. The first electron transfer reaction corresponds to the tyrosine Tyr10 amino acid residue oxidation, and the second to all three histidine (His6, His13 and His14) and one methionine (Met35) amino acid residues. The Aß peptides aggregation and amyloid fibril formation are electrochemically detected via the electroactive amino acids oxidation peak currents decrease that occurs in a time dependent manner. The Aß peptides redox behaviour is correlated with changes in the adsorption morphology from initially random coiled structures, corresponding to the Aß peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aß peptides in a ß-sheet configuration, observed by atomic force microscopy. Electrochemical studies of Aß peptides aggregation, mediated by the interaction with metal ions, particularly zinc, copper and iron, and different methodologies concerning the detection of Aß peptide biomarkers of AD in biological fluids, using electrochemical biosensors, are also discussed.

Amyloid Beta Peptide VHHQ, KLVFF, and IIGLMVGGVV Domains Involved in Fibrilization: AFM and Electrochemical Characterization.

The time-dependent structural modifications and oxidation behavior of specifically chosen five short amyloid beta (Aß) peptides, Aß1-16, Aß1-28, Aß10-20, Aß12-28, and Aß17-42, fragments of the complete human Aß1-40 peptide, were investigated by atomic force microscopy (AFM) and voltammetry. The objective was to determine the influence of different Aß domains (VHHQ that contains electroactive histidine H residues, KLVFF that is the peptide hydrophobic aggregation core, and IIGLMVGGVV that is the C-terminus hydrophobic region), and of Aß peptide hydrophobicity, in the fibrilization mechanism. The short Aß peptides absence of aggregation or the time-dependent aggregation mechanisms, at room temperature, in free chloride media, within the time window from 0 to 48 h, were established by AFM via changes in their adsorption morphology, and by differential pulse voltammetry, via modifications of the amino acid residues oxidation peak currents. The first oxidation peak was of tyrosine Y residue and the second peak was of histidine H and methionine M residues oxidation. A correlation between the presence of an intact highly hydrophobic KLVFF aggregation core and the time-dependent changes on the Aß peptides aggregation was found. The hydrophobic C-terminal domain IIGLMVGGVV, present in the Aß1-40 peptide, also contributed to accelerate the formation of Aß1-40 peptide aggregates and fibrils.

Pharmaceuticals released from senior residences: occurrence and risk evaluation.

One of the main pursuits, yet most difficult, in monitoring studies is to identify the sources of environmental pollution. In this study, we have identified health-care facilities from south European countries as an important source of pharmaceuticals in the environment. We have estimated that compounds consumed in by the elderly and released from effluents of senior residences can reach river waters at a concentration higher than 0.01 µg/L, which is the European Medicines Agency (EMA) threshold for risk evaluation of pharmaceuticals in surface waters. This study has been based on five health institutions in Portugal, Spain, and France, with 52 to 130 beds. We have compiled the pharmaceuticals dispensed on a daily base and calculated the consumption rates. From 54.9 to 1801 g of pharmaceuticals are consumed daily, with laxatives, analgesics, antiepileptics, antibiotics, and antidiabetic agents being the main drug families administered. According to excretion rates, dilution in the sewerage system, and elimination in wastewater treatment plants, macrogol, metformin, paracetamol, acetylcysteine, amoxicillin, and gabapentin, among others, are expected to reach river waters. Finally, we discuss the risk management actions related to the discharge of pharmaceuticals from senior residences to surface waters.

Alzheimer's disease amyloid beta peptides in vitro electrochemical oxidation.

The oxidative behaviour of the human amyloid beta (Aß1-40 and Aß1-42) peptides and a group of similar peptides: control inverse (Aß40-1 and Aß42-1), mutants (Aß1-40Phe10 and Aß1-40Nle35), rat Aß1-40Rat, and fragments (Aß1-28, Aß1-16, Aß10-20, Aß12-28, and Aß17-42), in solution or adsorbed, at a glassy carbon electrode, by cyclic and differential pulse voltammetry, were investigated and compared. Structurally the Aß1-40 and Aß1-42 sequences contain five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). The Aß peptide 3D structure influenced the exposure of the redox residues to the electrode surface and their oxidation peak currents. Depending on the amino acid sequence length and content, the Aß peptides gave one or two oxidation peaks. The first electron transfer reaction corresponded to the tyrosine amino acid residue oxidation, and the second to both histidines and methionine amino acid residues. The highest contribution to the second oxidation peak current was from His13, followed by His14 and His6 residues, and Met35 residue had the lowest contribution. The Aß peptides electron transfer depended on peptide hydrophobicity and 3D structure, the redox residues position in the sequence, the redox residues close to N-termini giving the highest oxidation peak currents.

Calcium-induced calmodulin conformational change. Electrochemical evaluation.

Calmodulin (CaM) is an essential protein present in all eukaryote cells, ranging from vertebrates to unicellular organisms. CaM is the most important Ca2+ signalling protein, composed of two domains, N- and C-terminal domains, linked by a flexible central α-helix, and is responsible for the regulation of numerous calcium-mediated signalling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. The oxidation mechanism of native and denatured CaM, at a glassy carbon electrode, was investigated using differential pulse voltammetry and electrochemical impedance spectroscopy. Native and denatured CaM presented only one oxidation peak, related to the tyrosine amino acid residue oxidation. Calcium-induced calmodulin conformational change and the influence of Ca2+ concentration on the electrochemical behaviour of CaM were evaluated, and significant differences, in the tyrosine amino acid residue peak potential and current, in the absence and in the presence of calcium ions, were observed. Gravimetric measurements were performed with a graphite coated piezoelectric quartz crystal with adsorbed CaM, and calcium aggregation by CaM was demonstrated.

Amyloid-ß peptides time-dependent structural modifications: AFM and voltammetric characterization.

The human amyloid beta (Aß) peptides, Aß1-40 and Aß1-42, structural modifications, from soluble monomers to fully formed fibrils through intermediate structures, were investigated, and the results were compared with those obtained for the inverse Aß40-1 and Aß42-1, mutant Aß1-40Phe(10) and Aß1-40Nle(35), and rat Aß1-40Rat peptide sequences. The aggregation was followed at a slow rate, in chloride free media and room temperature, and revealed to be a sequence-structure process, dependent on the physicochemical properties of each Aß peptide isoforms, and occurring at different rates and by different pathways. The fibrilization process was investigated by atomic force microscopy (AFM), via changes in the adsorption morphology from: (i) initially random coiled structures of ∼0.6 nm height, corresponding to the Aß peptide monomers in random coil or in α-helix conformations, to (ii) aggregates and protofibrils of 1.5-6.0 nm height and (iii) two types of fibrils, corresponding to the Aß peptide in a ß-sheet configuration. The reactivity of the carbon electrode surface was considered. The hydrophobic surface induced rapid changes of the Aß peptide conformations, and differences between the adsorbed fibrils, formed at the carbon surface (beaded, thin, <2.0 nm height) or in solution (long, smooth, thick, >2.0 nm height), were detected. Differential pulse voltammetry showed that, according to their primary structure, the Aß peptides undergo oxidation in one or two steps, the first step corresponding to the tyrosine amino acids oxidation, and the second one to the histidine and methionine amino acids oxidation. The fibrilization process was electrochemically detected via the decrease of the Aß peptide oxidation peak currents that occurred in a time dependent manner.

Flavonoids electrochemical detection in fruit extracts and total antioxidant capacity evaluation.

Phenolic compounds detection in fruit extracts from: açai, bacuri, buriti, blackberry, black mulberry, blueberry, juçara, physalis, raspberry, and tamarillo, have been performed by reverse-phase high performance liquid chromatography with electrochemical detection (RP-HPLC-EC), using two detectors in series: a wall-jet detector flow cell with a glassy carbon electrode, and a thin-layer flow cell detector with a boron doped diamond electrode. This methodology, in gradient elution mode, was successfully used to detect seventeen phenolic compounds in the fruit extracts. The total antioxidant capacity of the fruit extracts by the electrochemical quantitative index (EI) and the method of capture of diphenilpicrilhydrazil (DPPH(●)) free radical "efficient concentration" (EC50), was evaluated. A very good correlation between EI and EC50 assays has been obtained, the fruit with the highest total antioxidant capacity being blackberry, while physalis exhibited the lowest antioxidant power.

In situ electrochemical evaluation of dsDNA interaction with the anticancer drug danusertib nitrenium radical product using the DNA-electrochemical biosensor.

Danusertib is a kinase inhibitor and anti-cancer drug. The evaluation of the interaction between danusertib and dsDNA was investigated in bulk solution and using the dsDNA-electrochemical biosensor. The dsDNA-danusertib interaction occurs in two sequential steps. First, danusertib binds electrostatically todsDNA phosphate backbone through the positively charged piperazine moiety. The second step involved the pyrrolo-pyrazolemoiety and led to small morphological modifications in the dsDNA double helix which were electrochemically characterised through the changes of guanine and adenine residue oxidation peaks and confirmed by electrophoretic and spectrophotometric measurements. The nitrenium cation radical product of danusertib amino group oxidation was electrochemically generated in situ on the dsDNA-electrochemical biosensor surface. The danusertib nitrenium cation radical redox metabolite was covalently attached to the C8 of guanine residues preventing their oxidation. An interaction mechanism of dsDNA-danusertib is proposed and the formation of the danusertib redox nitrenium radical metabolite-guanine adduct explained.

In situ dsDNA-bevacizumab anticancer monoclonal antibody interaction electrochemical evaluation.

The interaction of the anticancer monoclonal antibody bevacizumab (BEVA) with double-stranded DNA (dsDNA) was studied by voltammetry and gel-electrophoresis in incubated samples and using the dsDNA-electrochemical biosensor. The voltammetric results revealed a decrease and disappearance of the dsDNA oxidation peaks with increasing incubation time, showing that BEVA binds to the dsDNA but no DNA oxidative damage was detected electrochemically. Non denaturing agarose gel-electrophoresis experiments were in agreement with the voltammetric results showing the formation of compact BEVA-dsDNA adduct. The dsDNA-electrochemical biosensor in incubated solutions showed that BEVA also undergoes structural modification upon binding dsDNA, and BEVA electroactive amino acid residues oxidation peaks were detected.

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences.

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na(+) and K(+) ion-containing solution, although only K(+) promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.

Electrochemical evaluation of glutathione S-transferase kinetic parameters.

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/µM and a LOD of 6.4µM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis-Menten and Lineweaver-Burk double reciprocal plots, was determined. A value of KM~100µM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60µmol/min per mg of GST.

Triazole-linked phenyl derivatives: redox mechanisms and in situ electrochemical evaluation of interaction with dsDNA.

The redox mechanism of two trisubstituted triazole-linked phenyl derivatives (CL41 and CL42) and a disubstituted triazole-linked phenyl derivative (CL2r50) were studied using cyclic, differential pulse and square wave voltammetry at a glassy carbon electrode. The CL41, CL42 and CL2r50 oxidation is a complex, pH-dependent irreversible process involving the formation of electroactive products that undergo two consecutive reversible oxidation reactions. The DNA interaction with CL41, CL42 and CL2r50 was investigated by differential pulse voltammetry using the dsDNA-electrochemical biosensor and in DNA/trisubstituted triazole incubated solutions. All three trisubstituted triazole-linked phenyl derivatives interacted with dsDNA causing morphological and oxidative damage to the dsDNA structure in a time-dependent manner. The DNA-electrochemical biosensor enabled the detection of oxidative damage to DNA following the occurrence of the 8-oxoGua and/or 2,8-oxoAde oxidation peaks.

In situ evaluation of gemcitabine-DNA interaction using a DNA-electrochemical biosensor.

The electrochemical behaviour of the cytosine nucleoside analogue and anti-cancer drug gemcitabine (GEM) was investigated at glassy carbon electrode, using cyclic, differential pulse and square wave voltammetry, in different pH supporting electrolytes, and no electrochemical redox process was observed. The evaluation of the interaction between GEM and DNA in incubated solutions and using the DNA-electrochemical biosensor was studied. The DNA structural modifications and damage were electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues and the occurrence of the free guanine residues electrochemical signal. The DNA-GEM interaction mechanism occurred in two sequential steps. The initial process was independent of the DNA sequence and led to the condensation/aggregation of the DNA strands, producing rigid structures, which favoured a second step, in which the guanine hydrogen atoms, participating in the C-G base pair, interacted with the GEM ribose moiety fluorine atoms.