Electrophile tocopheryl quinones from the phenolic antioxidants gamma-tocopherol and delta-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 gamma-tocopheryl quinone (gamma-TQ) form thiol adducts. The monoglutathion-S-yl derivative of gamma-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 delta-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 gamma-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. gamma-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.

Chromium(V)-mediated oxidative damage of deoxy-ribonucleic acids was investigated at neutral pH in aqueous solution by utilizing bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) (I) and bis(hydroxyethyl)-amino-tris(hydroxymethyl)methane)oxochromate(V) (II). Single-stranded and double-stranded (ds) calf thymus and human placenta DNA, as well as two oligomers, 5'-GATCTAGTAGGAGGACAAATAGTGTTTG-3' and 5'-GATCCAAGCAAACACTATTTGTCCTCCTACTA-3', were reacted with the chromium(V) complexes. Most products were separated and characterized by chroma-tographic and spectroscopic methods. Polyacrylamide gel electrophoresis experiments reveal more damage at G sites in comparison to other bases. Three primary oxidation products, 5-methylene-2-furanone (5-MF), furfural and 8-oxo-2'-deoxyguanosine, were characterized. A minor product, which appears to be thymine propenal, was also observed. The dsDNA produces more furfural than furanone. The formation of these two products resulted from hydrogen ion or hydride transfer from C1' and C5' positions of the ribose to the oxo-chromium(V) center. Since no enhancements of these products (except propenal) were observed in the presence of oxygen, mechanisms pertaining to the participation of activated oxygen species may be ruled out. The oxidation of the G base is most likely associated with an oxygen atom transfer from the oxo-metallates to the double bond between C8 and N7 of the purine ring. The formation of the propenal may be associated with an oxygen-activated species, since a marginal enhancement of this product was observed in the presence of oxygen. The formation of furfural in higher abundance over 5-MF for dsDNA was attributed to the ease of hydrogen ion (or hydride transfer) from the C5' compared to C1' position of the ribose within a Cr(V)-DNA intermediate in which the metal center is bound to the phosphate diester moiety.

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Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are converted to cytotoxic and carcinogenic metabolites, quinones, by detoxifying enzyme systems in animals. PAH metabolites such as the quinones can form Michael adducts with biological macromolecules containing reactive nucleophiles, making detection of exposure to PAHs difficult using conventional techniques. A technique has been developed for detecting exposure to PAHs. Tetramethylammonium hydroxide (TMAH) thermochemolysis coupled with GC/MS is proposed as an assay method for PAH quinones that have formed Michael adducts with biological molecules. Three PAH quinones (1,4-naphthoquinone, 1,2-naphthoquinone, and 1,4-anthraquinone) and 1,4-benzoquinone were reacted with cysteine, and the TMAH thermochemolysis method was used to assay for both thiol and amine adduction between the quinones and the cysteine. Additional studies with 1,4-naphthoquinone adducts to glutathione and bovine serum albumin showed the same thiol and amine TMAH thermochemolysis products with larger peptides as was observed with cysteine adducts. The TMAH GC/MS method clearly shows great promise for detecting PAH quinones, produced by enzymatic conversion of PAHs in biological systems, that have been converted to respective Michael adducts.