Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

Electrochemical Studies of Tirapazamine: Generation of the One-Electron Reduction Product

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono-and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.     

Electrochemical, UV--visible and EPR studies on nitrofurantoin: nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Electrochemical,UV-Visible and EPR studies on nitrofurantoin: Nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Reaction of the hypoxia-selective antitumor agent tirapazamine with a C1'-radical in single-stranded and double-stranded DNA: the drug and its metabolites can serve as surrogates for molecular oxygen in radical-mediated DNA damage reactions

The compound 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine; also known as SR4233, WIN 59075, and tirazone) is a clinically promising anticancer agent that selectively kills the oxygen-poor (hypoxic) cells found in tumors. When activated by one-electron enzymatic reduction, tirapazamine induces radical-mediated oxidative DNA strand cleavage. Using the ability to generate a single deoxyribose radical at a defined site in an oligonucleotide, we recently provided direct evidence that, in addition to initiating the formation of DNA radicals, tirapazamine can react with these radicals and convert them into base-labile lesions [Daniels et al. (1998) Chem. Res. Toxicol. 11, 1254-1257]. The rate constant for trapping of a C1'-radical in single-stranded DNA by tirapazamine was shown to be approximately 2 x 10(8) M(-1) s(-1), demonstrating that tirapazamine can substitute for molecular oxygen in radical-mediated DNA strand damage reactions. Because reactions of tirapazamine with DNA radicals may play an important role in its ability to damage DNA, we have further characterized the ability of the drug and its metabolites to convert a C1'-DNA radical into a base-labile lesion. We find that tirapazamine reacts with a C1'-radical in double-stranded DNA with a rate constant of 4.6 x 10(6) M(-1) s(-1). The mono-N-oxide (3) stemming from bioreductive metabolism of tirapazamine converts the C1'-radical to an alkaline-labile lesion more effectively than the parent drug. Compound 3 traps a C1'-radical in single-stranded DNA with a rate constant of 4.6 x 10(8) M(-1) s(-1) and in double-stranded DNA with a rate constant of 1.4 x 10(7) M(-)(1) s(-)(1). We have also examined the rate and mechanism of reactions between the C1'-radical and representatives from two known classes of "oxygen mimetic" agents: the nitroxyl radical 2,2,6, 6-tetramethylpiperidin-N-oxyl (4, TEMPO) and the nitroimidazole misonidazole (5). TEMPO traps the C1'-radical in single-stranded DNA (7.2 x 10(7) M(-1) s(-1)) approximately 3 times less effectively than tirapazamine, but 2 times as fast in double-stranded DNA (9.1 x 10(6) M(-1) s(-1)). Misonidazole traps the radical in single- (6. 9 x 10(8) M(-1) s(-1)) and double-stranded DNA (2.9 x 10(7) M(-1) s(-1)) with rate constants that are roughly comparable to those measured for the mono-N-oxide metabolite of tirapazamine. Finally, information regarding the chemical mechanism by which these compounds oxidize a monomeric C1'-nucleoside radical has been provided by product analysis and isotopic labeling studies.     

Electrochemical study of the reduction of toyocamycin and sangivamycin in aqueous media

The redox behaviour of two antibiotics, toyocamycin and sangivamycin, structurally related pyrrolopyrimidine nucleosides, and their reduction products in buffered aqueous media, have been examined by direct current polarography and cyclic voltammetry. Both compounds exhibit one 3-electron polarographic wave in the pH range 1-6. Macroscale electrolysis at the crest of the polarographic wave was followed electrochemically and by UV spectroscopy. The photochemical transformation of the reduction products on UV irradiation has been examined. It was found that the reduction of both compounds occurs in the pyrimidine ring, leading to two reduction products. One of these (lambda max = 306 nm) is photochemically reversible to the parent compound.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^-₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^-₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^-₂. Second order kinetics for the decay of RNO^-₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^-₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

Oxygen radical-mediated DNA damage by redox-active Cr(III) complexes.

The mechanism of DNA damage induced by Cr(III) complexes is currently unknown even though it is considered to be the ultimate biologically active oxidation state of chromium. In this study, we have employed the Salmonella reversion assay to identify mutagenic Cr(III) complexes. Cyclic voltammetry was used to differentiate the redox kinetics between mutagenic and selected nonmutagenic Cr(III) species. Plasmid relaxation of supercoiled DNA was employed to show in vitro interactions with plasmid DNA and correlate the interactions with the electrochemical behavior and biological activity. The results of this study demonstrate that the mutagenic Cr(III) complexes identified in the Salmonella reversion assay display characteristics of reversibility and positive shifts of the Cr(III)/Cr(II) redox couple consistent with the ability of these Cr(III) complexes to serve as cyclical electron donors in a Fenton-like reaction. These same mutagenic complexes display an ability to relax supercoiled DNA in vitro, presumably by the induction of single-strand breaks. Nonmutagenic complexes were selected to test different ligands to determine how the ligand directs the activity of Cr(III) complexes. All nonmutagenic complexes tested thus far have shown classical irreversibility, more negative reduction potentials, and an inability to relax supercoiled plasmid DNA. These results suggest that the mechanism by which chromium complexes potentiate mutagenesis involves an oxygen radical as an active intermediate. These data also demonstrate the effect of associated ligands with regard to the ability of a metal to generate an active redox center.     

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.     

DNA coated with cationic fullerene derivative. A possible microwire in water.

Fullerene derivative 2 carrying pyridinium cation bound to sonicated calf thymus DNA in water. The binding ratio was 1 fullerene unit to 1 phosphate residue, giving the complex where DNA strand is seemingly coated with electron-conducting fullerenes. Cyclic voltammetry shows three-step redox couples in the complex, and the current peaks were broadened and shifted to positive side as compared to uncomplexed 2. Binding of 2 onto grooves of DNA double helix was suggested.     

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl₃ (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)₃. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)₃ with ds-DNA. It was revealed that Yb(QS)₃ presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)₃ with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10⁻⁸ to 1.1 × 10⁻⁷ M. The interaction mode between Yb(QS)₃ and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^?₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^?₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^?₂. Second order kinetics for the decay of RNO^?₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^?₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

Electrochemical studies of η5-cyclopentadienyliron hexafluorophosphates of η6-substituted arenes and η6-heterocycles

Polarographic half-wave potentials for two reduction steps of 49 cyclopentadienyliron complexes of substituted arenes or heterocycles in dimethylformamide were determined. A fast cyclic voltammetry (10–40 V/s) was used to study the electron transfer kinetics of some of these complexes. After correction for the double layer effects, the rate constants of all the complexes studied show that the transfer of electrons in the second reduction step occurs significantly faster than in the first one. This was interpreted as a result of greater delocalization of electrons in the 20-electron complexes of iron compared to the 19-electron complexes.     

Template synthesis of novel carboxamide dinuclear copper (II) complex: spectral characterization and reactivity towards calf-thymus DNA

Dinuclear complexes Bis [aqua 1,8-(1,2-dicarboxamido benzene) 3,6-diazaoctane copper (II)/nickel (II)] tetrachloride (1 and 2) were synthesized by a two component one-pot metal template condensation between phthalic anhydride and 1,8-diamino 3,6-diazaoctane. Elemental analysis, molar conductance measurements, electronic absorption, infra-red, electron paramagnetic resonance, nuclear magnetic resonance, atomic absorption, and electron spray mass spectral studies have been performed to probe the nature and structure of the complexes. The interaction of copper (II) complex with calf thymus (CT-DNA) has been studied by using absorption, emission and circular dichoric spectral methods, viscometry, and cyclic voltammetry. A strong hyperchromism along with a red shift in UV bands and hypochromism in the ligand field band of the complex 1 on interaction with CT-DNA imply a covalent mode of DNA binding. This is further confirmed by studying the reactivity of complex 1 using circular dichroism and viscosity measurements. The variation in relative emission intensity of DNA-bound ethidium bromide observed upon treatment with the complex 1 parallel the trend of DNA binding studies. Cyclic voltammetry studies reveal that the complex 1 prefers to bind to DNA in Cu(II) rather than Cu(I) oxidation state.     

Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product

Mechanisms based on one-electron oxidation appear incomplete in explaining cellular radiosensitization by nitroaromatic compounds such as misonidazole. Evidence is presented for a novel mechanism that may be involved in enhancing DNA strand breakage due to a variety of agents, including ionizing radiation, that generate carbon-centered radicals on DNA deoxyribose. Under anaerobic conditions the carbon-centered radical generated selectively at C-5' of deoxyribose of thymidylate residues in DNA by the antitumor antibiotic neocarzinostatin reacts with misonidazole to produce a DNA damage product in the form of 3'-(formyl phosphate)-ended DNA. In an 18O-transfer experiment we find that the carbonyl oxygen of the activated formyl moiety (trapped as formyl-Tris) is derived from the nitro group oxygen of misonidazole. This result strongly supports a mechanism in which a nitroxide radical adduct, formed by the addition of misonidazole to the radical at C-5' of deoxyribose, cleaves between the N and O so as to form an oxy radical precursor of the formyl moiety and a two-electron reduction species of misonidazole.     

Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor

A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction.     

Synthesis and DNA binding studies of Mg(II) complex of Schiff base derived from vanillin and L-tryptophan

The Mg(II) complex of Schiff base (K[HL]) derived from vanillin and L-tryptophan could bind with herring sperm DNA. The binding behaviors between them in physiological pH environment (pH 7.40) have been studied by spectroscopy, cyclic voltammetry and viscosity methods. Binding ratios of n(Mg(II)): n(K[HL])?= 1:1 and n(Mg(II)L): n(DNA)?= 5:1 were confirmed. The obtained thermodynamic parameters suggest that the interaction between Mg(II)L and DNA is driven mainly by entropy. Combined with fluorimeteric studies, cyclic voltammetry, CD spectroscopy and viscosity methods, the results indicate the interaction modes between Mg(II)L and DNA are mainly with intercalation and involving some electrostatic interaction.     

Some aspects of lead(II) DNA interactions

The interaction of Pb(II) ions with calf-thymus DNA was studied by differential pulse polarography, sweep voltammetry, cyclic voltammetry, chromatography on hydroxyapatite and viscosity measurements. Pb(II) ions may interact with nucleic acid via phosphate groups causing some stabilization of the DNA structure. However, the more specific interaction occurs with nucleic bases. The latter interaction destabilizes the nucleic acid structure and leads to inter- and intra-chain binding.     

Voltammetric investigation of DNA damage induced by nitrofurazone and short-lived nitro-radicals with the use of an electrochemical DNA biosensor

Electrochemical behavior of nitrofurazone (NFZ) was investigated with the use of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. The pH-dependence of NFZ was studied at a glassy carbon electrode (GCE) in ethanol/Britton-Robinson buffer (30:70), and short-lived nitro-radicals were generated by the reduction of NFZ at high pHs (>7.0). In the presence of DNA, the DPV peak current of NFZ decreased and the peak potential shifted negatively, which indicated that there was an electrostatic interaction between NFZ and DNA. An electrochemical dsDNA/GCE biosensor was prepared to study the DNA damage produced in the presence NFZ; this process was followed with the use of the Co(phen)(3)(2+) electroactive probe. Also, the oxidation peaks of guanosine (750mV) and adenosine (980mV) indicated that DNA damage was related directly to the nitro-radicals. Experiments demonstrated that DNA damage occurred via two different steps while NFZ was metabolized and nitro-radicals were produced. Novel work with AFM on the NFZ/DNA interaction supported the suggestion that in vivo, the nitro-radicals were more cytotoxic than the NFZ molecules. A linear DPV calibration plot was obtained for NFZ analysis at a modified dsDNA/GCE (concentration range: 2.50×10(-6)-3.75×10(-5)molL(-1); limit of detection: 8.0×10(-7)molL(-1)), and NFZ was determined successfully in pharmaceutical samples.