Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase.
We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.
D000632:Aminopyrine / Q000494:pharmacology; D000693:Anaerobiosis; D000818:Animals; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000494:pharmacology*; D007213:Indomethacin / Q000494:pharmacology; D008297:Male; D010100:Oxygen / Q000494:pharmacology; D010544:Peroxidases / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000494:pharmacology*; D012756:Sheep; D020058:Styrene; D013343:Styrenes / Q000378:metabolism*
D003497:Cyclic N-Oxides; D005609:Free Radicals; D013343:Styrenes; D000632:Aminopyrine; D020058:Styrene; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D005978:Glutathione; D010100:Oxygen; D007213:Indomethacin