Milan Mazúr

Role of oxygen radicals in DNA damage and cancer incidence.

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction—oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC → TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, β-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed (Mol Cell Biochem 266: 37–56, 2004)

Copper(II) complexes with derivatives of salen and tetrahydrosalen: a spectroscopic, electrochemical and structural study

Abstract Salen type complexes, CuL, the corresponding tetrahydrosalen type complexes, Cu[H4]L, and N,N′-dimethylated tetrahydrosalen type complexes, Cu[H2Me2]L, were investigated using cyclic voltammetry, and electronic and ESR spectroscopy. In addition, the analogous copper(II) complexes with a derivative of the tetradentate ligand ‘salphen’ [salphen=H2salphen=N,N′-disalicylidene-1,2-diaminobenzene] were studied. Solutions of CuL, Cu[H4]L and Cu[H2Me2]L are air-stable at ambient temperature, except for the complex Cu(tBu, Me)[H4]salphen [H2(tBu, Me)[H4]salphen=N,N′-bis(2-hydroxy-3-tert-butyl-5-methylbenzyl)-1,2-diaminobenzene]. Cu(tBu, Me)[H4]salphen interacts with dioxygen and the ligand is oxidatively dehydrogenated (–CH2–NH–→–CN–) to form Cu(tBu, Me)[H2]salphen and finally, in the presence of base, Cu(tBu, Me)salphen. X-ray structure analysis of Cu(tBu, Me)[H2Me2]salen confirms a slightly tetrahedrally distorted planar geometry of the CuN2O2 coordination core. The complexes were subjected to spectrophotometric titration with pyridine, to determine the equilibrium constants for adduct formation. It was found that the metal center in the complexes studied is only of weak Lewis acidity. In dichlormethane, the oxidation Cu(II)/Cu(III) is quasireversible for the CuL type complexes, but irreversible for the Cu[H4]L and Cu[H2Me2]L type. A poorly defined wave was observed for the irreversible reduction Cu(II)/Cu(I) at potentials less than −1.0 V. The ESR spectra of CuL at both 77 K and room temperature reveal that very well resolved lines can be attributed to the interaction of an unpaired electron spin with the copper nuclear spin, 14N donor nuclei and to a distant interaction with two equivalent protons [∣ACu(iso)∣≈253 MHz, ∣AN(iso)∣≈43 MHz, ∣AN(iso)∣≈20 MHz]. These protons are attached to the carbon atoms adjacent to the 14N nuclei. In contrast to CuL, the number of lines in the spectra of the complexes Cu[H4]L and Cu[H2Me2]L is greatly reduced. At room temperature, only a quintet with a considerably smaller nitrogen shf splitting constant [∣AN(iso)∣≈27 MHz] is observed. Both factors, planarity and conjugation, are thus essential for the observation of distant hydrogen shf splitting in CuL. Due to the CN bond hydrogenation, the coordination polyhedra of the complexes Cu[H4]L and Cu[H2Me2]L is more flexible and more sensitive to ligand modification than that of CuL. The electron-withdrawing effect of the phenyl ring of the phenylenediamine bridge is reflected in a reduction of the copper hyperfine coupling constants in Cu(tBu, Me)[H4]salphen and Cu(tBu, Me)[H2Me2]salphen complexes [∣ACu(iso)∣≈215 MHz].

Oxygen free radical generating mechanisms in the colon: do the semiquinones of vitamin K play a role in the aetiology of colon cancer?

It is proposed that bile acids (deoxycholic acid), the K vitamins, iron(II) complexes and oxygen interact to induce an oncogenic effect in the colon by the generation of free radicals. In the relatively low oxidising/reducing conditions of the colonic lumen the K vitamins exist in the reduced form; however, if absorbed into the mucosa they have the capacity to be chemically oxidised and to enter into a redox cycle yielding oxygen radicals. The semiquinone radical of K(1) (phylloquinone) has been stabilised in bile acid mixed micelles and investigated by electron paramagnetic resonance spectroscopy and quantum chemical calculations. The estimated half-life of the radical was about 30 min which confirms a remarkably high stability in aqueous micellar solution. A model is presented in which the reduced K vitamins may initiate superoxide radical, O2(-*) generation leading to Fe(II) mediated Fenton reactions in the stem colon cells.

Quantitative electron paramagnetic resonance (EPR) spectrometry with a TE104 double rectangular cavity Part 1. A simple alignment procedure for the precision positioning of the sample

Abstract The Bruker TE104 double rectangular cavity is commonly utilized for quantitative electron paramagnetic resonance (EPR) spectrometric measurements. The sample, together with an appropriate standard, can be studied simultaneously within such a cavity under identical states of machine tuning and parameter settings. However, even under such conditions, there still remain sources of error arising from sample size, shape and positioning of the sample tube within the cavity. In this paper such sources of error have been investigated and a procedure to minimize the errors by precision insertion of the sample tubes within the TE104 cavity is described.

Effect of hydrogenation on electronic and distant magnetic properties in copper(II) complexes with derivatives of tetrahydrosalen and salen. X-ray crystal structure of [Cu{Bu,Me(saltmen)}] complex

Abstract The salen complex CuL and the corresponding tetrahydrosalen complex Cu[H4]L were investigated by ESR spectroscopy and molecular orbital calculations [H2L = N,N′-bis(3-tert-butyl-5-methylsalicylidene)-2,3-diamino-2,3-dimethylbutane; H2[H4]L = N,N′-bis(2-hydroxy-3-tert-butyl-5-methylbenzyl)-2,3-diamino-2,3-dimethylbutane]. X-ray structure determination of CuL confirmed a slightly distorted planar geometry of the CuN2O2 coordination core. The ESR spectra of CuL at both 78 K and room temperature revealed that very well resolved lines cannot be attributed to the interaction with copper nuclear spin and 14N donor nuclei alone. Computer simulation showed that in addition to copper hyperfine (giso = 2.094, |ACu(iso)| = 270 MHz, room temperature) and nitrogen superhyperfine structure [|AN(iso)| = 46 MHz] a distant interaction with two equivalent protons is also present [|AH(iso)| = 23 MHz]. These protons are attached to the carbon atoms adjacent to 14N nuclei. In contrast the number of lines in the spectrum of the hydrogenated analogue Cu[H4]L is greatly reduced. At room temperature only a quintet with considerable smaller nitrogen shf constant [|AN(iso)|] = 25 MHz is observed. Thus, both factors planarity and conjugation, are essential for the observation of distant hydrogen shf splitting in CuL. The ESR findings are in good agreement with calculated spin densities by QR-INDO/1 method.

Quantitative electron paramagnetic resonance (EPR) spectrometry with a TE104 double rectangular cavity Part 2. Analysis of sample and TE104 cavity error sources associated with the movement of line-like samples into the TE104 cavity

Abstract In this study the sample- and double TE 104 cavity-associated error sources have been analysed. It was found that in quantitative EPR experiments, the EPR signal intensity of identical full-length cavity line-like samples, which were packed by the same procedure with the same powdered material into identical sample tubes, then precision inserted into the same positions within the double TE 104 cavity using a special sample insertion procedure, and measured using the same instrumental parameters, could be obtained with experimental errors of about 3–5%. However, if the procedures for packing and inserting the powdered samples into the cavity were not used, the associated experimental errors could be over 20%. The errors associated with the movement of a full-length cavity line-like sample (30 mm length) into the double TE 104 cavity have been analysed. This analysis has shown that the experimental dependence of the EPR signal intensity upon the movement of the sample along the x -axis of the double TE 104 cavity exhibited a 12.5 mm wide plateau over which the EPR signal intensity remained constant within an experimental error 0.5–0.8%. Because the centre of the plateau coincided with the situation in which centre of the sample was positioned in the cavity centre, this position is recommended for quantitative EPR measurements. The existence of the plateau in the case of full-length cavity line-like samples was found to be the principal difference when compared with the corresponding dependence for point-like samples.

A study of copper(II) carboxylato complexes with the biological ligands nicotinamide and papaverine

The X-band Electron Spin Resonance spectra, ESR, of Cu(II) complexes with the carboxylic acids, HCOOH, CH3COOH, ClCH2COOH, Cl2CHCOOH, Cl3CCOOH, F2ClCOOH, CH3CH2COOH, niflumic acid and the ligands nicotinamide and papaverine have been studied. The compounds form either mononuclear or binuclear structures which can be qualitatively correlated with the strengths of the acid and base. Analysis of the spin Hamiltonian parameters for the monomeric species indicate that the molecule has nearly axial symmetry ðgk . g’Þ with the unpaired electron in the dx2 –y 2 orbital. In many cases the perpendicular feature is sufficiently well resolved showing superhyperfine features due to the nitrogen ligand nuclei. Ak varies over the range 130 ! 188 G and the variation appears to arise as a consequence of changes in gk: The dimeric species show ESR spectra typical of Cu(II)-carboxylate complexes which consist of 3 (4 if E – 0) lines. These are assigned to Bz1; Bx2;y2 and Bz2 since the Q-band spectra of similar molecules indicate that the Bx1;y1 transition(s) lies below Bz2 which is itself near zero field. The observed seven-line hyperfine coupling on the Bz1 transition of some of the dimeric complexes is due to the two equivalent copper nuclei ðI ¼ I1 þ I2 ¼ 3Þ: The singlet – triplet separation has been estimated from the spin Hamiltonian parameters to give values of l2Jl in the range of approximately 220– 290 cm 21 which are typical of the compounds of this type.

Self-assembly of hydrogen-bonded supramolecular structures of two copper(II) 2-bromobenzoate complexes with 4-pyridylmethanol and nicotinamide

The structures of [Cu(2-Brbz)2(4PM)2(H2O)] (1) and [Cu(2-Brbz)2(NIA)2] · 2H2O 2 [where 2-Brbz is the 2-bromobenzoate anion, 4-PM is the 4-pyridylmethanol and NIA is nicotinamide] have been determined by X-ray and characterized by EPR spectroscopy. The Cu2+ cation in 1 is coordinated by a pair of oxygens from monodentate 2-bromobenzoate anions by a pair of pyridine nitrogens from monodentate 4-pyridylmethanol ligands and finally by a water forming a tetragonal-pyramidal coordination polyhedron. The Cu2+ cation in 2 is coordinated by two pairs of oxygens from the asymmetric bidentate 2-bromobenzoate anions and by a pair of pyridine nitrogen atoms from the monodentate nicotinamide in trans positions, forming an extremely elongated bipyramid. The molecules of both complexes are linked by O–H ··· O, C–H ··· O and for 2 by N–H ··· O hydrogen bonds, which create three-dimensional hydrogen-bonding networks. EPR spectra of 1 and 2 are in agreement with X-ray data. Nicotinamide as well as 4-pyridylmethanol are suitable ligands for construction of hydrogen bonding coordination polymers.

Electron paramagnetic resonance signal intensity of a line-like sample with a variable length situated at an arbitrary position along the common sample-cavity axis. Theoretical prediction versus experimental measurement

The integral equations used for the theoretical prediction of the relative EPR-signal intensity of variable length line-like samples situated at the central position of a microwave cavity have been computed numerically for the cases where: (i) the microwave field is non-uniform but the modulation field is uniform, and (ii) the microwave and modulation fields are non-uniform. In both cases, stress is placed on the variable length of the sample and the length of the active part of the microwave cavity. The theoretically predicted and experimentally observed electron paramagnetic resonance (EPR) signal intensity are in a very good agreement for sample lengths from 1.3 to 50 mm. However, for the cases where the sample length is greater than that of the cavity, the theoretical equations and experimental results can only be reconciled if the cavity is assumed to have an active length, *a=40 mm, considerably greater than the actual length of the cavity, a=23.5 mm. Comparison of the theoretically predicted and experimentally obtained EPR-signal intensities indicate that those parts of the sample situated in the upper and lower sample access holes of the rectangular cavity can contribute significantly to the measured signal intensity. The possible influence of this phenomenon as a significant source of error in quantitative EPR measurements is discussed. The integral equations have been generalised to describe the signal intensity for line-like sample positioned at any point along the common sample-cavity axis.

EPR Spectroscopy of a Clinically Active (1:2) Copper(II)-Histidine Complex Used in the Treatment of Menkes Disease: A Fourier Transform Analysis of a Fluid CW-EPR Spectrum

Redox active transition metal ions (e.g., iron and copper) have been implicated in the etiology of many oxidative stress-related diseases including also neurodegenerative disorders. Unbound copper can catalyze formation of reactive oxygen species (hydroxyl radicals) via Fenton reaction/Haber–Weiss chemistry and therefore, under physiological conditions, free copper is potentially toxic and very rarely exists inside cells. Copper(II) bound to the aminoacid l-histidine represents a species discovered in blood in the mid 60s and since then extensive research on this complex was carried out. Copper bound to l-histidine represents an exchangeable pool of copper(II) in equilibrium with the most abundant blood plasma protein, human serum albumin. The structure of this complex, in aqueous solution, has been a subject of many studies and reviews, however without convincing success. The significance of the (1:2) copper(II)-l-histidine complex at physiological pH documents its therapeutic applications in the treatment of Menkes disease and more recently in the treatment of infantile hypertrophic cardioencephalomyopathy. While recently the (1:2) Cu(II)-l-His complex has been successfully crystallized and the crystal structure was solved by X-ray diffraction, the structure of the complex in fluid solution at physiological pH is not satisfactorily known. The aim of this paper is to study the (1:2) Cu(II)-l-histidine complex at low temperatures by X-band and S-band EPR spectroscopy and at physiological pH at room temperature by Fourier transform CW-EPR spectroscopy.