We studied
nitric oxide-mediated protection against
tert-butyl hydroperoxide (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 containing the human iNOS gene (K/VP. 5-iNOS). K/VP.5-iNOS cells were 2-fold less 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]diazen-1- ium-1, 2-diolate), protected K/VP.5 cells against t-BuOOH-induced cytotoxicity and provided an additional increment of protection in K/VP.5-iNOS cells. Under conditions of excess t-BuOOH and deficiency of
iron catalytic sites (
hemoglobin, Hb) in K/VP.5-iNOS cells, the increase of intracellular Hb concentration is the main contributor to enhanced sensitivity of the cells 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 resulted in the production of dinitrosyl complexes of non-
heme iron and hexacoordinated
heme iron nitrosyl complexes based on low-temperature EPR spectra. 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-iNOS cells. Upon addition of
NOC-15 to K/VP.5-iNOS cells, the EPR signal of dinitrosyl complexes of non-
heme iron was enhanced, and the EPR signal of nitrosylated
heme iron became discernible. It was determined that levels of non-
heme and
heme (
hemoglobin)
iron were dramatically decreased in K/VP.5-iNOS cells compared to K/VP.5 cells, thus explaining the decreased intensities of EPR signals of nitrosylated species. In addition, t-BuOOH-induced oxoferryl-Hb-associated
protein-centered
free radical species as well as t-BuO(*) alkoxyl radicals were observed in these two cell lines. These t-BuOOH-induced 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-expressing cells. Most importantly, the combined action of
NOC-15 and t-BuOOH resulted in complete elimination of both oxoferryl-associated radical EPR signals as well as those from dinitrosyl complexes of non-
heme iron and nitrosylated
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 oxide-mediated protection against t-BuOOH-induced cytotoxicity. First, prolonged endogenous production of
nitric oxide results in a decreased content of catalytic non-
heme iron and
heme iron sites through posttranscriptional regulation of
iron homeostasis. Second,
nitric oxide can chemically reduce t-BuOOH-induced oxoferryl and t-BuO(*) alkoxyl radicals.