DNA alkylation by carbon-centered radicals.

1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals.

Formation of methyl radicals during the catalase-mediated oxidation of formaldehyde hydrazone.

It has been suggested that formaldehyde hydrazone, a condensation product of hydrazine with formaldehyde, plays an important role in hydrazine-promoted DNA methylation in vivo. The present study demonstrated by spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide and tert-nitrosobutane that catalase-mediated oxidation of formaldehyde hydrazone generates methyl radicals. Both the use of two spin-traps and parallel studies of oxygen consumption were important for excluding possible artefacts of spin-trapping experiments with tert-nitrosobutane.

Comparative studies of nifurtimox uptake and metabolism by drug-resistant and susceptible strains of Trypanosoma cruzi.

1. Nifurtimox uptake and metabolism by epimastigote forms of three strains of Trypanosoma cruzi (Basileu, Y, YuYu) with different drug responsiveness in mice experimental infections were compared. 2.

Iron(III) binding in DNA solutions: complex formation and catalytic activity in the oxidation of hydrazine derivatives.

A strong interaction between iron(III) and calf thymus DNA at pH 7.4 was demonstrated in the present study by separation of the complex by column chromatography and by the slow kinetics of iron(III) removal from DNA by disodium-1,2-dihydroxybenzene-3,5-disulfonate (Tiron). An equilibrium constant of 2.

Formation of 8-methylguanine as a result of DNA alkylation by methyl radicals generated during horseradish peroxidase-catalyzed oxidation of methylhydrazine.

Methylhydrazine oxidation promoted by horseradish peroxidase-H2O2 or ferricyanide led to the generation of high yields of methyl radicals and to the formation of 7-methylguanine and 8-methylguanine upon interaction with calf thymus DNA. Methyl radicals were identified by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and tert-nitrosobutane. The methylated guanine products were identified in the neutral hydrolysates of treated DNA by high pressure liquid chromatography (HPLC) analysis and spiking with authentic samples.

ESR detection of free radical intermediates during autoxidation of 5-hydroxyprimaquine.

Autoxidation of 5-hydroxyprimaquine, a putative metabolite of the antimalarial primaquine, was studied by oxygen consumption and ESR spectroscopy. 5-Hydroxyprimaquine underwent fast autoxidation under mild conditions (pH 7.4-8.

Restricted bioreductive metabolism of a nitroimidazole-thiadiazole derivative with curative action in experimental Trypanosoma cruzi infections.

The bioreductive activation of megazol [2-amino-5(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole] promoted by ferredoxin: NADP+ oxidoreductase, rat liver microsomes and cellular fractions of Trypanosoma cruzi, Y strain, was investigated. Direct ESR detection and characterization by computer simulation of the megazol nitro anion radical were possible in the presence of NADPH and ferredoxin: NADP+ oxidoreductase under anaerobic conditions. By contrast, the megazol nitro anion radical was not detected in the presence of either rat liver microsomes or cellular fractions of T.

Free radical generation during delta-aminolevulinic acid autoxidation: induction by hemoglobin and connections with porphyrinpathies.

delta-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and saturnism, undergoes autoxidation leading to ammonium ion and probably the corresponding alpha-ketoaldehyde. This reaction is accelerated by addition of oxyhemoglobin (oxyHb) and other iron complexes. OxyHb is concomitantly oxidized to metHb; the apparent second-order rate constant of oxyHb/ALA coupled oxidation is ca.

DNA alterations induced by the carbon-centered radical derived from the oxidation of 2-phenylethylhydrazine.

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethylhydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 mM phenelzine.

Hemoglobin-mediated oxidation of the carcinogen 1,2-dimethylhydrazine to methyl radicals.

Oxidation of 1,2-dimethylhydrazine (SDMH) catalyzed by hemoglobin is investigated by oxygen consumption studies, ESR spin-trapping experiments, and gas chromatography. Kinetic analysis and the study of the effects of superoxide dismutase, catalase, and azide on reaction rates indicate that SDMH oxidation is primarily dependent on ferric hemoglobin and autoxidatively formed H2O2. SDMH oxidation generates both methyl and hydroxyl radicals as ascertained by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide, and tert-nitrosobutane.

Direct ESR detection of a free radical intermediate during the peroxidase-catalyzed oxidation of the antimalarial drug primaquine.

Oxidation of the antimalarial primaquine by horseradish peroxidase and H2O2 was demonstrated by visible light absorption and ESR spectroscopy. Initial product analysis indicated a 15% yield of O-demethoxylation products, methanol and the quinone-imine derivative, and organic extractable polymeric material. Horseradish peroxidase was substituted by methemoglobin, and both enzymes showed greater activity at acidic pH values.

Enzymatic activation of the carcinogens 2-phenylethylhydrazine and 1,2-dimethylhydrazine to carbon-centered radicals.

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylhydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase - H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylhydrazine oxidation generates ten times more carbon-centered radicals than 1,2-dimethylhydrazine oxidation. This result is in agreement with oxygen consumption studies from which the apparent KM values of 8.

Primaquine can mediate hydroxyl radical generation by Trypanosoma cruzi extracts.

Primaquine increases the NAD(P)H dependent oxygen consumption and hydroxyl radical generation by extracts of Trypanosoma cruzi, the causative agent of Chagas' disease. Spin-trapping studies show that hydroxyl radical yield is completely inhibited by catalase and slightly increased by SOD, indicating that radical generation is dependent on the pair primaquine-NAD(P)H and their interaction product, H2O2. Primaquine effects upon Trypanosoma cruzi extracts are compared with those obtained with nifurtimox, a compound effective in the treatment of Chagas' disease.

Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes.

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al.

Spin-trapping of methyl radical in the oxidative metabolism of 1,2-dimethylhydrazine.

A carbon-centered free radical formed during oxidative metabolism of 1,2-dimethylhydrazine has been spin-trapped with alpha-(4-pyridyl-1-oxide)N-tert-butyl nitrone and 2-methyl-2-nitrosopropane. In the horseradish peroxidase/H2O2 catalyzed oxidation, the trapped species was identified as the methyl radical by the characteristic 1:3:3:1 quartet pattern of the 2-methyl-2-nitroso propane adduct. A carbon-centered radical is also formed during microsomal oxidation of 1,2-dimethylhydrazine in the presence of NADPH.

DNA strand scission by the carbon radical derived from 2-phenyl-ethylhydrazine metabolism.

Oxyhemoglobin catalyzed oxidation of the tranquilizing drug 2- phenylethylhydrazine induces single strand breaks (nicks) in the supercoiled pBR322 plasmid DNA. Spin-trapping studies have established a clear correlation between 2-phenylethyl radical yield and the DNA strand scission activity observed during 2- phenylethylhydrazine oxidation. The same correlation is obtained in the presence of active oxygen species scavengers or when the carbon radical is generated under anaerobic conditions by ferricyanide oxidation of the drug.

Carbon radicals in the metabolism of alkyl hydrazines.

The metabolism of phenelzine (2-phenylethylhydrazine) by rat liver microsomes yields phenylacetaldehyde, 2-phenylethanol, and ethylbenzene. A carbon radical is formed during the oxidative metabolism of phenelzine that reacts with the prosthetic heme of cytochrome P-450 and irreversibly inactivates the enzyme. The radical has been spin-trapped, isolated, and shown by mass spectrometry to be the 2-phenylethyl radical.

The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation.

Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives.

N-Phenylprotoporphyrin IX formation in the hemoglobin-phenylhydrazine reaction. Evidence for a protein-stabilized iron-phenyl intermediate.

Hemoglobin-catalyzed phenylhydrazine oxidation, a model for Heinz-body hemolytic anemias, is known to result in hemoglobin degradation and erythrocyte lysis. The catalytic reaction is shown here to be terminated by inactivation of the prosthetic heme groups after each heme moiety catalyzes the consumption of 6 oxygen molecules and 6 phenylhydrazines, and the formation of 5 benzenes. The phenyl residue not converted to benzene is primarily found, after acidic methanol workup, covalently bound to a nitrogen of protoporphyrin IX.

Hemin-catalyzed generation of triplet acetone.

Hemin can substitute for horseradish peroxidase as a catalyst for the aerobic oxidation of isobutanal to acetone and formate. Previous studies have shown that the chemiphosphorescent emission observed in the enzyme-catalyzed reaction is due to the production of acetone in its triplet state. Although no chemiphosphorescence is observed with the model system (hemin), generation of triplet acetone in this system is indicated by an analysis of data for energy transfer to the 9,10-dibromoanthracene-2-sulfonate ion and for interception of the excited species by the sorbate ion, a known triplet quencher.