Electrophile tocopheryl quinones in apoptosis and mutagenesis: Thermochemolysis of thiol adducts with proteins and in cells

Electrophile tocopheryl quinones from the phenolic antioxidants γ-tocopherol and δ-tocopherol form Michael adducts with the thiol nucleophile glutathione. These tocopheryl quinones are involved in cytotoxicity, apoptosis, and mutagenesis, and their biologic properties are associated with the depletion of intracellular thiols. We now show that both proteins and tissues treated with the electrophile γ-tocopheryl quinone (γ-TQ) form thiol adducts. The monoglutathion-S-yl derivative of γ-TQ was subjected to thermochemolysis with the strong methylating base tetramethylammonium hydroxide. GC/MS showed four signature peaks and a fragmentation pattern characteristic of the thiol adduct. Similarly, pure monoglutathion-S-yl and diglutathion-S-yl derivatives of δ-TQ were subjected to thermochemolysis, and GC/MS showed characteristic fragmentation patterns for thiol adducts. The four signature peaks were identified when pure proteins with accessible thiol groups (hemoglobin and histone), FBS, and tissue culture medium and cell preparations were treated with γ-TQ. Signature peaks in both complete medium and washed cells showed the presence of both soluble and insoluble thiol adducts. The effective or free arylating electrophile concentration in complete medium should always be evaluated in tissue culture studies. γ-TQ is a mutagen but not a genotoxin; therefore, the histone adduct may be a previously unrecognized histone modification involved in chromatin dynamics leading to mutagenesis.