Mechanisms of nitric oxide protection against tert-butyl hydroperoxide-induced cytotoxicity in iNOS-transduced human erythroleukemia cells

We studied nitric oxide-mediated protection against tert-butyl hydroperoxid e (t-BuOOH)induced cytotoxicity in a subline of human erythroleukemia K562 cells (K/VP.5) and in K/VP.5 cells transduced with a retroviral vector cont aining the human iNOS gene (K/VP.5-iNOS). K/VP.5-iNOS cells were 2-fold les s sensitive to the cytotoxic effects of t-BuOOH compared to K/VP.5 cells. A nitric oxide donor, NOC-15 ((Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]d iazen-1-ium-1,2-diolate), protected K/VP.5 cells against t-BuOOH-induced cy totoxicity and provided an additional increment of protection in K/VP.5-iNO S cells. Under conditions of excess t-BuOOH and deficiency of iron catalyti c sites (hemoglobin, Hb) in K/VP.5-iNOS cells, the increase of intracellula r Hb concentration is the main contributor to enhanced sensitivity of the c ells to t-BuOOH-induced cytotoxicity (despite the effects of small amounts of endogenously produced nitric oxide). Protection against t-BuOOH-induced cytotoxicity in K/VP.5-iNOS cells was diminished by treatment with an iNOS inhibitor, L-N-G-monomethylarginine (L-NMA), but was restored upon addition of NOC-15 to L-NMA-treated cells. Incubation of K/VP.5 cells with NOC-15 r esulted in the production of dinitrosyl complexes of non-heme iron and hexa coordinated heme iron nitrosyl complexes based on low-temperature EPR spect ra. In K/VP.5-iNOS cells, only a weak EPR signal of dinitrosyl complexes of non-heme iron was observed in the absence of NOC-15. In addition, no heme iron nitrosyl complexes were discernible in the EPR spectra from K/VP.5-iNO S cells. Upon addition of NOC-15 to K/VP.5-iNOS cells, the EPR signal of di nitrosyl complexes of non-heme iron was enhanced, and the EPR signal of nit rosylated heme iron became discernible. It was determined that levels of no n-heme and heme (hemoglobin) iron were dramatically decreased in K/VP.5-iNO S cells compared to K/VP.5 cells, thus explaining the decreased intensities of EPR signals of nitrosylated species. In addition, t-BuOOH-induced oxofe rryl-Hb-associated protein-centered free radical species as well as t-BuO. alkoxyl radicals were observed in these two cell lines. These t-BuOOH-induc ed radical species were greatly reduced in K/VP.5-iNOS cells compared to K/ VP.5 cells, consistent with a reduction in heme iron levels in the iNOS-exp ressing cells. Most importantly, the combined action of NOC-15 and t-BuOOH resulted in complete elimination of both oxoferryl-associated radical EPR s ignals as well as those from dinitrosyl complexes of non-heme iron and nitr osylated heme iron in both K/VP.5-iNOS cells and K/VP.5 cells. We conclude that two independent pathways operate in erythroleukemia cells for nitric o xide-mediated protection against t-BuOOH-induced cytotoxicity. First, prolo nged endogenous production of nitric oxide results in a decreased content o f catalytic non-heme iron and heme iron sites through posttranscriptional r egulation of iron homeostasis. Second, nitric oxide can chemically reduce t -BuOOH-induced oxoferryl and t-BuO. alkoxyl radicals.