metallothionein and 5-5-dimethyl-1-pyrroline-1-oxide

Topics: Animals; Anions; Brain; Cells, Cultured; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Hydroxyl Radical; Metallothionein; Mice; Mitochondria; Neuroprotective Agents; Oxidopamine; Protein Isoforms; Spin Labels; Superoxides

A variety of Cu reconstituted metallothioneins (MTs) containing different amounts of copper ions together with Cd7-MT free of copper were prepared and used in spin trapping experiments designed to show cadmium is not a Fenton active metal. A significant increase of the DMPO/.OH adduct was observed, with increased concentrations of the copper containing MTs, H2O2 enhanced DMPO/.OH adduct formation, catalase and the Cu (I) specific chelating agent bathocuproine, reduced DMPO/.OH adduct formation. These results suggest that Cu (I) and H2O2 both have important roles in the production of active species in these systems and cause DMPO/.OH formation. However, Cd7-MT showed no ability to cause generation of DMPO adducts with H2O2 seeming to indicate cadmium is not a Fenton metal. To test this hypothesis further trapping studies were run with added sulphite and lipid peroxide using both commercial MT and Cd7-MT since cadmium causes peroxidation in vivo. Commercial MT generates radicals with added sulphite and peroxide, Cd7-MT does not, demonstrating that cadmium is not a Fenton metal. These results help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.

Topics: Cadmium; Copper; Cyclic N-Oxides; DNA Damage; Dose-Response Relationship, Drug; Drug Interactions; Electron Spin Resonance Spectroscopy; Hydroxyl Radical; Lipid Peroxidation; Metallothionein; Reproducibility of Results

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyl-piperidine- N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7, 8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.

Topics: Antioxidants; Chromans; Cyclic N-Oxides; Cysteine; Dimethyl Sulfoxide; Electron Spin Resonance Spectroscopy; Ethanol; Free Radical Scavengers; Free Radicals; Glutathione; Hydroxyl Radical; Mercaptoethanol; Metallothionein; Nitrogen Oxides; Phenols; Sulfhydryl Compounds