bromochloroacetic-acid and 5-5-dimethyl-1-pyrroline-1-oxide

We report here the application of the electron spin resonance technique to detect free radicals formed by the hydroperoxidase activity of prostaglandin H synthase in cells. Studies were done using keratinocytes obtained from hairless mice. These cells can be prepared in large number and possess significant prostaglandin H synthase activity. Initial attempts to directly detect free radical metabolites of several amines in cells were unsuccessful. A technique was developed based on the ability of some free radicals formed by prostaglandin hydroperoxidase to oxidize reduced glutathione (GSH) to a thiyl radical, which was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Phenol and aminopyrine are excellent hydroperoxidase substrates for this purpose and thus were used for all further experiments. Using this approach we detected the DMPO/GS.thiyl radical adduct catalyzed by cellular prostaglandin hydroperoxidase. The formation of the radical was dependent on the addition of substrate, inhibited by indomethacin, and supported by either exogenous arachidonic acid or endogenous arachidonic acid released from phospholipid stores by Ca2+ ionophore A-23187. The addition of GSH significantly increased the intracellular GSH concentration and concomitantly stimulated the formation of the DMPO/GS.thiyl radical adduct. Phenol, but not aminopyrine, enhanced thiyl radical adduct formation and prostaglandin formation with keratinocytes while both cofactors were equally effective in incubations containing microsomes prepared from keratinocytes. These results suggest that prostaglandin hydroperoxidase-dependent co-oxidation of chemicals can result in the intracellular formation of free radical metabolites.

Topics: Aminopyrine; Animals; Arachidonic Acid; Arachidonic Acids; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Epidermis; Female; Free Radicals; Glutathione; Indomethacin; Keratins; Male; Mice; Mice, Hairless; Microsomes; Oxidation-Reduction; Peroxidases; Phenol; Phenols; Prostaglandin-Endoperoxide Synthases; Seminal Vesicles; Sheep; Spin Labels

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.

Topics: Animals; Benzene Derivatives; Carcinogens; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Epidermis; Free Radicals; Keratins; Mice; Peroxides; tert-Butylhydroperoxide