Detection of glutathione thiyl free radical catalyzed by prostaglandin H synthase present in keratinocytes. Study of co-oxidation in a cellular system

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.