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 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.
Yalowich
J C
JC
Department of Pharmacology, University of Pittsburgh School of Medicine, Graduate School of Public Health, Pennsylvania 15238, USA. Yalowich+@pitt.edu
Gorbunov
N V
NV
Kozlov
A V
AV
Allan
W
W
Kagan
V E
VE
D000438:Alcohols / Q000378:metabolism; D001384:Azetidines / Q000494:pharmacology; D004357:Drug Synergism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D005615:Freezing; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D020014:K562 Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D012190:Retroviridae / Q000235:genetics; D020122:tert-Butylhydroperoxide / Q000378:metabolism / Q000633:toxicity*
C111679:1-(N-(3-ammoniopropyl)-N-(n-propyl)amino)diazen-1-ium-1,2-diolate; D000438:Alcohols; D001384:Azetidines; D005609:Free Radicals; D006454:Hemoglobins; D020030:Nitric Oxide Donors; C059688:alkoxyl radical; D009569:Nitric Oxide; D020122:tert-Butylhydroperoxide; D007501:Iron; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II