DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.
Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.
Olinski
R
R
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Nackerdien
Z
Z
Dizdaroglu
M
M
D002460:Cell Line; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005609:Free Radicals; D005720:Gamma Rays; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006878:Hydroxides; D017665:Hydroxyl Radical; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D009363:Neoplasm Proteins / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D013941:Thymine; D014443:Tyrosine
D002843:Chromatin; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D005609:Free Radicals; D006878:Hydroxides; D009363:Neoplasm Proteins; D017665:Hydroxyl Radical; D014443:Tyrosine; D006861:Hydrogen Peroxide; D013941:Thymine