Tumour promoter tert-butyl-hydroperoxide induces peroxynitrite formation in human erythrocytes.
In the present study we demonstrated that human erythrocytes under normal conditions release small amounts of peroxynitrite (ONOO-) that can be considerably increased by the tumour promoter t-butylhydroperoxide (t-BHP), with a subsequent increase in lipid peroxidation and inhibition of Ca2+ pump ATPase activity. By causing oxidative stress in human erythrocytes with t-BHP, ONOO- release was increased approximately ten fold. N-monomethyl-L-arginine (L-NMMA) inhibited ONOO- release by approximately 90% while D-NMMA had no effect. The interaction of t-BHP with hemoglobin (Hb) and methemoglobin (MetHb) caused the production of superoxide (O2-) and hydrogen peroxide (H2O2). The differential direct effect of t-BHP on Hb and MetHb was investigated by taking their spectra in the presence or absence of cytochrome C. Erythrocyte membranes treated either with t-BHP or with ONOO- were subjected to oxidative stress with a subsequent increase in malondialdehyde (MDA) production and decrease in membrane fluidity, as estimated by the fluorescence polarization of 1,6,diphenyl-1,3,5-hexatriene (DPH). Ca2+ pump ATPase activity was decreased in t-BHP and/or ONOO-(-)treated erythrocytes, indicating that the subsequent intracellular calcium increase promoted ONOO- production by activating the Ca(2+)-calmodulin-dependent NO-synthase activity. These results suggest that hemoglobin oxidation by the tumour promoters play a key role in oxidative damage to erythrocytes and that the t-BHP/ Hb redox system could be a useful tool for investigating the tumour promoting efficacy of organic hydroperoxides.
Department of Physiology, University of Athens, Medical School, Greece.
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G
Deliconstantinos
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V
Villiotou
J C
JC
Stavrides
D003574:Cytochrome c Group / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000502:physiology; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008163:Luminescent Measurements; D008315:Malondialdehyde / Q000378:metabolism; D008560:Membrane Fluidity; D008706:Methemoglobin / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010545:Peroxides / Q000494:pharmacology*; D019323:omega-N-Methylarginine / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
D003574:Cytochrome c Group; D005609:Free Radicals; D006454:Hemoglobins; D009566:Nitrates; D010545:Peroxides; C039642:peroxynitric acid; D019323:omega-N-Methylarginine; D008315:Malondialdehyde; D008706:Methemoglobin; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D010100:Oxygen