Peroxidase-mediated formation of glutathione conjugates from polycyclic aromatic dihydrodiols and insecticides

Using two peroxidative systems (prostaglandin H synthase/arachidonic acid and horseradish peroxidase/H2O2) we observed GSH conjugate formation with a number of compounds including polycyclic aromatic hydrocarbon-diols (PAH-diols), insecticides, and steroids. Several of the conjugates were characterized by chromatography, uv-vis spectrophotometry, and FAB mass spectroscopy. Conjugate formation is dependent upon a functioning peroxidase, GSH, and is markedly enhanced (3- to 10-fold) by the inclusion of a number of reducing cosubstrates including phenol, uric acid, phenylbutazone, and acetaminophen. The mechanism of conjugate formation appears to involve addition of thiyl radical to alkene bonds conjugated to an electron releasing group probably by resonance stabilization of the carbon-centered radical intermediate. Thiyl radicals are formed either directly by GSH reduction of the peroxidase or indirectly by GSH reduction of radicals formed from reducing cosubstrates. The nitrone spin trap, 5,5-dimethyl-1-pyrroline N-oxide, which traps thiyl radicals, totally inhibits production of GSH conjugates in both peroxidative systems. Conjugation of PAH-diols, some of which are penultimate carcinogens, would prevent their metabolism to the diol-epoxides, an ultimate carcinogenic species of PAH. Conjugation by peroxidases appears to be a general pathway for glutathione conjugate formation that may lead to potential detoxification of chemicals.