The C242T CYBA polymorphism as a major determinant of NADPH oxidase activity in patients with cardiovascular disease.

Single nucleotide polymorphisms (SNP) in the CYBA gene encoding p22(phox) have been associated with respiratory burst and cardiovascular phenotypes. We previously reported a reduced phagocytic respiratory burst activity in healthy adults with the C242T SNP, but found no correlation between CYBA SNPs and coronary artery disease (CAD) phenotype. Using lymphoblastoid cells, we hypothesized that CYBA SNPs affect enzyme activity in patients with cardiovascular disease (CVD), but would not be associated with angiographic severity of CAD due to confounding by risk factors. We established lymphoblastoid cell lines from patients with CVD and genotyped the study cohort for CYBA SNPs and phenotyped each subject's coronary angiogram for CAD severity. As quantified by electron spin resonance, superoxide production in picomoles per 10(6) resting lymphoblastoid cells per minute for the CC, CT, and TT genotypes of the C242T SNP were 16.2+/-1.4, n=70, 11.9+/-0.7, n=87, and 11.9+/-1.5, n=28, respectively (P=0.002). The -930(A/G) and A640G SNPs did not affect superoxide production (P > 0.2). Expression of p22(phox) was not affected as determined by real-time RT-PCR and Western blot analysis. The C242T CYBA SNP is associated with altered NADPH oxidase activity in lymphoblastoid cells of patients with CVD. By reducing the influence of confounding environmental factors, lymphoblastoid cell lines could serve as a tool to assess direct genotype/phenotype interactions of candidate genes known to affect atherosclerosis.

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts.

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Inherited enhancement of hydroxyl radical generation and lipid peroxidation in the S strain rats results in DNA rearrangements, degenerative diseases, and premature aging.

Previously by selection and inbreeding of Wistar rats susceptible or resistant to the cataractogenic effect of galactose the S and R rat strains differing in the intensity of hexose transport into the animal cells were developed. High level of OH-radical generation and enhanced lipid peroxidation are revealed in the liver and myocardium of the S rats in contrast to the R rats. Data are obtained supporting the view that enhanced generation of OH-radicals within the S rat tissues is due to oxidation and autooxidation of the abundant amounts of monosacharides intensely accumulating in the rat cells. In spite of continuous inbreeding for more than 40 generations and a high rate of homozygosity, numerous DNA rearrangements are revealed in the S rat genomes. Fragility of the S rat cell membranes is detected. Cataracts and other lens lesions, emphysema, tumors, cardiomyopathy-like changes in the myocardium, scoliosis, brain disfunctions are characteristic of the S rats, as well as low fertility and short life-span indicative of premature aging.

Role of quinone-iron(III) interaction in NADPH-dependent enzymatic generation of hydroxyl radicals.

To study the effect of chelation of iron ions by quinones on the generation of OH radicals in biological redox systems, we have synthesized quinones that can form complexes with Fe(III) ions: 2-phenyl-4-(butylamino)naphtho[2,3-h]quinoline-7,12-dione (Qbc) and 2-phenyl-4-(octylamino)naphtho[2,3-h]quinoline-7,12-dione (Qoc). A quinone with a similar structure without chelating group was synthesized as a control sample: 2-phenyl-5-nitronaphtho[2,3-g]indole-6,11-dione (Qn). Using optical spectroscopy, we determined the stability constant of Qbc with Fe(III) [Ks = (7 +/- 1) x 10(18) M-3] and the stoichiometry of the complex Fe(Qbc)3 in chloroform solutions. One-electron reduction potentials of Qbc, Qn, and adriamycin in dimethyl sulfoxide were measured by cyclic voltammetry. In the presence of Fe(III) the one-electron reduction potentials shifted toward positive values by 0.16 and 0.1 V for Qbc and adriamycin, respectively. Using the spin trap 5,5'-dimethyl-1-pyroline N-oxide (DMPO) and EPR, it was found that Qbc in the Fe(III) complex stimulated the formation of OH radicals in the enzymatic system consisting of NADPH and NADPH-cytochrome P-450 reductase more efficiently than adriamycin and quinone Qn. This is indicated by the absence of a lag period in the spin adduct appearance for Qbc and by a significantly higher rate of the spin adduct production, as well as by a larger absolute concentration of the spin adduct obtained for Qbc in comparison with Qn in the presence of Fe(III).(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxyl radical generation by oligonucleotide derivatives of anthracycline antibiotic and synthetic quinone.

For the first time the covalent binding of anticancer anthracycline drugs and their potential synthetic analogs to oligonucleotides of different sequences is proposed for obtaining site-specific DNA scission in systems in vitro and in vivo. New compounds such as daunomycin (Dm) and synthetic naphthoquinone (NQ), covalently bound to the heptadeoxynucleotide of pCCAAACA (Dm-pN7) and decadeoxythymidilate (pT10p-NQ), have been obtained. These oligonucleotide derivatives can form specific complexes with complementary oligonucleotide sequences; these compounds and their complementary complexes can be reduced by purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), it has been shown that in aerobic conditions Dm-pN7 and pT10p-NQ are capable of generating OH radicals with and without complementary oligonucleotides. The chemical stability of the compounds in redox reactions has been studied. Oligonucleotide derivatives of natural and synthetic quinones capable of generating OH radicals seem to be a promising tool for site-specific scission of DNA in solution and in cells.