Effect of the Copper- 1,10-Phenanthroline Complex on Hep-G2 Cells

Abstract

Interaction of heme-and non-heme iron with H202 or organic hydroperoxides is known to produce oxidative stress due to formation of oxofenyl-and oxygen-derived free radical species. Using low-temperature EPR, we demonstrated oxoferrylhemoglobin (oxoferryl-Hb) free radical species in human erythroleukemia W P . 5 cells (K562-derived etoposide-resistant subline) upon exposure to tert-butylhydroperoxide (t-BuOOH). The intensity of oxoferryl EPR signals was proportional to intracellular Hb concentrations (which were manipulated by incubating the cells with different concentrations of hemin for 24 hr), and was correlated with the cell mortality, and the amount of oxidative stress. The latter was quantitated by a novel technique based on metabolic integration of cis-parinaric acid into major classes of membrane phospholipids and their subsequent fluorescence-HPLC assay. Preincubation of the cells with an NOdonor, NOC-15, resulted in the formation of non-heme iron nitrosyl complexes as well as hexa-(R-state) and pentacoordinate (T-state) Hb-nitrosyl complexes. Nitrosylation of nonheme iron and Hb prevented t-BuOOH-induced: (i) generation of oxofenyl free radical species, (ii) oxidative stress, and (iii) cell death. Importantly, KNP.5 cells transfected with the human inducible NO synthase (iNOS) gene were also resistant to t-BuOOH-induced oxidative stress. Since NO did not protect against oxidative stress induced by a lipophillic azo-initiator of peroxyl radicals (AMVN), we conclude that protective effects of NO were due to the formation of iron-nitrosyl complexes rather than scavenging of peroxyl radicals.Pharmacology,