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BackgroundSoman, a potent irreversible acetylcholinesterase (AChE) inhibitor, induces delayed neuronal injury by reactive oxygen species (ROS). Midazolam is used in patients with pathologic effects of oxidative stresses such as infection, hemodynamic instability and hypoxia. We investigated whether midazolam protects the Central Nervous System (CNS) from soman intoxication. The present study was performed to determine whether midazolam protects B35 cells from ROS stress for the purpose of exploring an application of midazolam to soman intoxication.MethodsGlucose oxidase (GOX) induced ROS stress was used in a B35 neuroblastoma cell model of ROS induced neuronal injury. To investigate the effect of midazolam on cell viability, LDH assays and fluorescence activated cell sorting (FACS) analysis was performed. Western blotting was used for evaluating whether Akt-phosphorylation is involved in cell-protective effects of midazolam.ResultsGOX derived ROS injury decreased cell viability about 1.6-2 times compared to control; midazolam treatment (5 and 10 µg/ml) dose-dependently increased cell viability during ROS injury. On western blots, Akt-phosphorylation was induced during pretreatment with midazolam; it was diminished during co-treatment with LY-294002, an inhibitor of Akt-phosphorylation. FACS analysis confirmed that the cell protective effect of midazolam is mediated by an anti-apoptotic effect. GOX-induced apoptosis was inhibited by midazolam and the finding was diminished by LY-294002.ConclusionsMidazolam protects neuronal cells from GOX-induced ROS injury; this effect is mediated by an anti-apoptotic effect through Akt-phosphorylation. This shows that midazolam may be useful in soman intoxication.

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1’s activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair.

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1’s activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair. [ABSTRACT FROM AUTHOR]

Background: Soman, a potent irreversible acetylcholinesterase (AChE) inhibitor, induces delayed neuronal injury by reactive oxygen species (ROS). Midazolam is used in patients with pathologic effects of oxidative stresses such as infection, hemodynamic instability and hypoxia. We investigated whether midazolam protects the Central Nervous System (CNS) from soman intoxication. The present study was performed to determine whether midazolam protects B35 cells from ROS stress for the purpose of exploring an application of midazolam to soman intoxication., Methods: Glucose oxidase (GOX) induced ROS stress was used in a B35 neuroblastoma cell model of ROS induced neuronal injury. To investigate the effect of midazolam on cell viability, LDH assays and fluorescence activated cell sorting (FACS) analysis was performed. Western blotting was used for evaluating whether Akt-phosphorylation is involved in cell-protective effects of midazolam., Results: GOX derived ROS injury decreased cell viability about 1.6-2 times compared to control; midazolam treatment (5 and 10 µg/ml) dose-dependently increased cell viability during ROS injury. On western blots, Akt-phosphorylation was induced during pretreatment with midazolam; it was diminished during co-treatment with LY-294002, an inhibitor of Akt-phosphorylation. FACS analysis confirmed that the cell protective effect of midazolam is mediated by an anti-apoptotic effect. GOX-induced apoptosis was inhibited by midazolam and the finding was diminished by LY-294002., Conclusions: Midazolam protects neuronal cells from GOX-induced ROS injury; this effect is mediated by an anti-apoptotic effect through Akt-phosphorylation. This shows that midazolam may be useful in soman intoxication.