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.