Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.
The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
Marek
M
Rác
Department of Immunology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15, Olomouc, Czech Republic.
Michal
M
Křupka
Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15, Olomouc, Czech Republic.
Svatopluk
S
Binder
Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
Michaela
M
Sedlářová
Department of Pharmacology and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15, Olomouc, Czech Republic.
Zuzana
Z
Matušková
Department of Immunology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15, Olomouc, Czech Republic.
Milan
M
Raška
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic.
Pavel
P
Pospíšil
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D020552:Comet Assay; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D017665:Hydroxyl Radical / Q000633:toxicity*; D007501:Iron / Q000633:toxicity; D007938:Leukemia / Q000378:metabolism / Q000473:pathology; D015227:Lipid Peroxidation / Q000187:drug effects; D008315:Malondialdehyde / Q000032:analysis; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000187:drug effects*; D050050:Protein Carbonylation / Q000187:drug effects; D026082:Singlet Oxygen / Q000378:metabolism*
C045076:Fenton's reagent; D026082:Singlet Oxygen; D017665:Hydroxyl Radical; D008315:Malondialdehyde; D006861:Hydrogen Peroxide; D007501:Iron