Oxidative Stress leads to Altered Signaling

Reference 

Experiment Description 

Result 

Azimzadeh et al., 2017 

In vitro. HCAECs were irradiated with 0.5 Gy of X-rays (0.5 Gy/min). Protein carbonylation and GSTO1 antioxidant levels were measured with a carbonylation assay and immunoblotting, respectively. Proteins from various signaling pathways including RhoGDI, p16, and p21 were measured with immunoblotting. 

After 0.5 Gy, carbonyl content increased a maximum of 1.2-fold and GSTO1 decreased a maximum of 0.78-fold. After 0.5 Gy, p-RhoGDI decreased a maximum of 0.7-fold, p16 increased a maximum of 1.5-fold, and p21 increased a maximum of 1.2-fold. 

Kenchegowda et al., 2018 

In vivo. Male 3- to 5-month-old Gottingen minipigs and Sinclair minipigs were whole-body irradiated with 1.7-2.3 Gy of ⁶⁰Co gamma rays (0.6 Gy/min). Both survivors (n=23) and euthanized moribund animals (n=17) had measurements taken for oxidative stress and altered signaling taken from the heart. SOD, CAT, and p67 (subunit of NADPH oxidase/NOX, involved in producing superoxide) levels were determined with western blot. ELISA and western blot were used to measure altered signaling in the IGF/PI3K/Akt pathway. 

Compared to survivors, radiation induced a 2.1-fold increase in p67, 0.87-fold decrease in SOD, and a 0.83-fold decrease in CAT (non-significant, ns) in the deceased group. Compared to survivors, the ratio of activated (phosphorylated) to total IGF-1R and the ratio of activated (phosphorylated) to total Akt both decreased 0.5-fold in the deceased group. 

Kook et al., 2015 

In vitro. MC3T3-E1 osteoblast-like cells were irradiated with 2, 4, and 8 Gy of X-rays (1.5 Gy/min). ROS were measured with a fluorescent probe, and SOD, CAT, and GSH antioxidant activities were determined with assay kits. Protein levels in the Nrf2/HO-1 signaling pathway were determined by either western blot or RT-PCR. 

ROS increased linearly at 2 and 4 Gy up to 1.4-fold at 8 Gy (significant changes at 4 Gy and 8 Gy). GSH and SOD were decreased 0.7-fold at 4 Gy and 0.5-fold at 8 Gy (insignificant increases at 2 Gy). CAT was also decreased but not significantly. HO-1 increased 3.3-fold after 4 Gy and 4.9-fold after 8 Gy (insignificant increase at 2 Gy). Nrf2 increased 2.3-fold after 8 Gy. Runx2 mRNA was decreased 0.5-fold after 8 Gy. 

Bai et al., 2020 

In vitro. Rat-derived bone marrow-derived mesenchymal stem cells (bmMSCs) were irradiated with 2, 5, and 10 Gy of ¹³⁷Cs gamma rays. Mitochondrial and cellular ROS levels were determined with fluorescent probes. RT-qPCR was performed to measure antioxidant enzyme expression. Protein expression in various signaling pathways was measured by western blot. 

Mitochondrial ROS increased 1.6-fold at 2 Gy (non-significant), 2-fold at 5 Gy, and 2.3-fold at 10 Gy. Cellular ROS increased 1.2-fold at 2 Gy, 1.5-fold at 5 Gy, and 2.1-fold at 10 Gy. Antioxidants SOD1, SOD2, and CAT all decreased about 0.9-fold (ns for SOD2) after 2 Gy, 0.8- to 0.7-fold at 5 Gy, and 0.7- to 0.4-fold at 10 Gy. Runx2 decreased 0.9-fold at 2 and 5 Gy and 0.6-fold at 10 Gy. p21 increased 1.6-fold at 5 Gy and 2.5-fold at 10 Gy. p53 increased 1.6-fold at 5 Gy and 1.7-fold at 10 Gy. p16 remained unchanged. 

Fan et al., 2017 

In vivo. 10-week-old male C57BL/6J mice were irradiated with ⁶⁰Co gamma rays at 3 Gy/day for 5 days. Left ventricular cardiac tissue was harvested for analysis. ROS was detected by dihydroethidium (DHE) staining. MAPK and Nrf2 signaling molecules were measured by western blot. 

Following irradiation, ROS production increased by 3.6-fold. 

p-p38/p38 decreased by 0.36-fold and p-Nrf2/Nrf2 decreased by 0.14-fold. 

Hasan, Radwan & Galal, 2020 

In vivo. 6-week-old male Wistar rats were irradiated with 6 Gy ¹³⁷Cs gamma rays. Oxidative stress was measured by MDA and GSH in heart tissue. Angiotensin II (AngII) and aldosterone, key molecules in the RAAS pathway, were measured with ELISA kits in serum. 

Following irradiation, MDA levels increased by 1.5-fold and GSH levels decreased by 0.5-fold. AngII and aldosterone increased 1.4-fold compared to control. 

Azimzadeh et al., 2015 

In vivo. Male 10-week-old C57BL/6 mice were irradiated with 8 and 16 Gy of X-rays. SOD, MDA, and protein carbonylation levels were determined with immunoblotting, lipid peroxidation, and protein carbonylation assays, respectively, in heart tissue. Proteins in various signaling pathways were measured with immunoblotting in heart tissue. 

SOD decreased 0.7-fold at both 8 and 16 Gy and MDA increased 1.4-fold after 8 Gy and 2.1-fold after 16 Gy. Protein carbonylation increased 1.4-fold after 16 Gy. Levels and activity of proteins in the PI3K/Akt pathway were decreased between 0.5- and 0.1-fold at both 8 and 16 Gy. The ERK/MAPK pathway was found decreased 0.5-fold at 16 Gy and the p38/MAPK pathway was found increased 1.3-fold at 16 Gy. p16 was increased 1.6-fold at both 8 and 16 Gy. p21 was increased 2.4-fold at both 8 and 16 Gy. 

Sakata et al., 2015 

In vitro. HUVECs were irradiated with 10 Gy X-rays at a dose rate of 5 Gy/min. Measurements were performed 0-72 h post-irradiation. ROS were detected by fluorescence microscopy. MAPK, Akt, p-p38, JNK and ERK1/2 signaling molecules were measured by western blot. 

Following 10 Gy irradiation, the intensity representing ROS generation increased 20- and 30-fold at the 24 and 72 h timepoints, respectively. 

MAPK, p38 and JNK remained unchanged for the 72 h measured following 10 Gy irradiation.  

p-Akt/Akt in HUVECs after 10 Gy irradiation showed an initial decrease at 5 min and a delayed decrease of 0.5-fold at 6-24 h. p-ERK1/2 decreased at 5 min then increased to a maximum 1.75-fold change. 

Wortel et al., 2019 

In vitro. BAECs were irradiated with 10 Gy ¹³⁷Cs gamma rays at a rate of 1.66 Gy/min. Extracellular H2O2 was measured by Amplex Red Assay, intracellular H₂O₂ levels were determined by HyPer sensor and peroxynitrite was quantified by chemiluminescence assay. Superoxide levels were quantified by luminescence after treatment with Diogenes Complete Enhancer Solution. The activation of the ASMase enzyme and the levels of ceramide were quantified by radioenzymatic assay to deter mine the changes on the ASMase/ceramide pathway. 

Following 10 Gy irradiation, intracellular H₂O₂ increased to a maximum 1.35-fold. Extracellular H₂O₂ increased by 1.75-fold. Peroxynitrite increased by 2.86-fold after 10 Gy (Fig 5). Superoxide levels increased over 350% at 2 minutes after 10 Gy irradiation. ASMase activity increased to a maximum 5.6-fold at 5 min after irradiation, then decreased and remained unchanged until the 30 min time-point. Ceramide increased from -500 to over 3000 pmol/106 cells. The significance of these changes was not indicated against a control. 

Azimzadeh et al., 2021 

In vivo. Male C57BL/6J mice 8 weeks of age were irradiated with 16 Gy of X-rays to the heart. SOD antioxidant activity and MDA in heart tissue were determined with an assay kit and lipid peroxidation assay, respectively. The level of proteins in MAPK pathways were determined by ELISA in heart tissue. 

After 16 Gy, SOD decreased 0.8-fold and MDA increased 1.3-fold. After 16 Gy, p-ERK increased 1.5-fold, p-p38 increased 1.3-fold, and p-JNK increased 1.3-fold. 

Xin et al., 2015 

In vitro and in vivo. 

In vitro. MC3T3-E1 osteoblast-like cells were exposed to microgravity for 96 hours. ROS were determined with a fluorescent probe and the RANK/RANKL pathway was measured using RANKL and OPG assay kits. 

In vivo. Male 8-week-old Sprague-Dawley rats were exposed to hind-limb suspension for 6 weeks. Femur and plasma MDA and femur sulfhydryl levels were measured with assay kits and the RANK/RANKL pathway was measured in the femur using RANKL and OPG assay kits. 

In vitro. ROS increased 1.5-fold and the RANKL/OPG ratio increased 1.6-fold. 

In vivo. Serum and femur MDA increased 1.4-fold and femur sulfhydryl decreased 0.6-fold. The RANKL/OPG ratio increased 3.5-fold. 

Sun et al., 2013 

In vitro. MC3T3-E1 osteoblast-like cells were exposed to microgravity (0.01G) for 96 hours. ROS production was measured by a fluorescent probe. The RANKL/OPG ratio was determined by assay kit and Runx2 mRNA expression was determined by RT-qPCR 

ROS increased 1.5-fold. The RANKL/OPG ratio increased 1.6-fold. Runx2 expression decreased 0.4-fold. 

Yoo, Han & Kim, 2016 

In vitro. Preosteoblast MC3T3-E1 cells were exposed to microgravity conditions by a 3D clinostat at a speed from 1-10 rpm. Oxidative stress was measured by Cu/Zn-SOD, Mn-SOD and catalase activity. Signaling molecules, p-Akt, phosphorylation of the mechanistic target of rapamycin p-(mTOR), and p-ERK were measured by western blot. 

Following microgravity exposure, Cu/Zn-SOD and Mn-SOD levels decreased by 0.24 and 0.65-fold, respectively. Signaling molecules p-Akt decreased by 0.36-fold. p-mTOR and p-ERK decreased by 0.58-fold. 

Diao et al., 2018 

In vivo. The left femur of rats was studied after exposure to simulated microgravity. Oxidative stress was measured by MDA, SOD, and CAT levels. RANK/RANKL signaling pathway was measured in rat femur by enzyme-linked immunoassay detection of OPG/RANKL molecules. Signaling molecule, Runx2, mRNA levels were measured by quantitative real time PCR. The Wnt/β-catenin pathway was measured by western blot for β-catenin protein levels. 

MDA increased by 1.4-fold. SOD and CAT levels decreased by 0.4-fold. OPG/RANKL decreased by 0.6-fold. Runx2 mRNA levels decreased 0.04-fold (Fig 8d). β-catenin decreased 0.6-fold. 

El-Missiry et al., 2018 

In vivo. Male Wistar rats were irradiated with gamma rays (¹³⁷Cs source, 4 Gy, 0.695 cGy/s) and measurements were taken from the hippocampus. Assay kits were used to assess levels of oxidative stress for marker 4-HNE (4-hydroxy-2-nonenal) and antioxidant markers GSH, glutathione peroxidase (GPx) and glutathione reductase (GR). Levels of p53 were determined using an assay kit. 

After 4 Gy, 4-HNE increased 2.4-fold, protein carbonylation increased 3.2-fold, GSH decreased 0.4-fold, GPx decreased 0.3-fold, GR decreased 0.2-fold, and p53 increased 2.7-fold. 

Suman et al., 2013 

In vivo. Female adult C57BL/6J mice were irradiated with 1.6 Gy of ⁵⁶Fe or 2 Gy of ¹³⁷Cs gamma irradiation at 1 Gy/min, then measurements were taken from the cerebral cortex. ROS levels were determined with flow cytometry and 4-HNE levels were assessed with immunohistochemical staining. p21 and p53 levels were determined with immunoblotting. 

ROS increased a maximum of 1.2-fold after gamma rays and 1.4-fold after ⁵⁶Fe radiation. The number of 4-HNE+ cells increased a maximum of 4.4-fold after gamma radiation and 14-fold after ⁵⁶Fe radiation. p21 increased a maximum of 1.5-fold after gamma rays and 3-fold after ⁵⁶Fe radiation. p53 increased a maximum of 8.4-fold after gamma rays and 9-fold after ⁵⁶Fe radiation. 

Limoli et al., 2004 

In vivo. Adult male C57BL/6J mice were irradiated with 1-10 Gy of X-ray at 1.75 Gy/min. MDA levels in the hippocampus were measured using an assay kit and western blot was used to determine p53 and p21 levels. 

In vitro. Neural precursor cells from the rat hippocampus were irradiated with 1-10 Gy of X-ray at 4.5 Gy/min. ROS levels were measured using CM-H2DCFDA dye and Western blot was used to measure p53 and p21 levels. 

MDA levels increased about 30% at 10 Gy. ROS increased a maximum of 31% at 1 Gy and 35% at 5 Gy, after 24 and 12 hours, respectively. At 5 Gy, p53 levels increased a maximum of 4-fold, while p- p21 also increased at this dose. 

Tian et al., 2020 

In vivo. C57BL/6J mice (including miR-137-/- and Src-/- models) underwent middle cerebral artery occlusion (MCAO) to simulate ischemic stroke and measurements were taken 7 days later in the cerebral cortex. ROS levels were measured with DCFH-DA fluorescent dye. Signaling molecules were measured with western blotting or RT-qPCR. 

ROS increased 1.8-fold. ERK1/2, p38 and JNK mRNA increased 2- to 3- fold. The ratios of phosphorylated to total ERK1/2, p38 and JNK increased 2- to 3- fold as well. 

Hladik et al., 2020 

In vivo. Female B6C3F1 mice were exposed to total body ⁶⁰Co gamma irradiation at 0.063, 0.125, or 0.5 Gy and at a dose rate of 0.063 Gy/min. Measurements from the hippocampus were taken up to 24 months post-irradiation. Protein levels in various signaling pathways (CREB, p38, ERK1/2, pro-apoptotic Bax and cleaved caspase 3, anti-apoptotic Bcl-xL) were determined with immunoblotting. 

Carbonylated proteins (indicative of ROS levels) were elevated in the 0.125 and 0.5 Gy group by approximately 25% and 30%, respectively. CREB phosphorylation increased by approximately 20% and 25% at 0.063 and 0.125 Gy, respectively. Phosphorylated p38 increased by approximately 100% and 80% at 0.063 and 0.125 Gy, respectively. Phosphorylated ERK1/2 increased by approximately 100% and 90% at 0.063 and 0.125 Gy, respectively.  

Anti-apoptotic BCL-xL decreased by 1.7-fold at 0.5 Gy, whereas pro-apoptotic Bax increased by approximately 2-fold at this dose. Caspase 3 also increased by approximately 2-fold at 0.5 Gy. 

Carvour et al., 2008 

In vitro. Mesencephalic dopaminergic neuronal cell line (N27) derived from rat mesencephalon were exposed to 3, 10, or 30 μM of H₂O₂. ROS levels were detected using dihydroethidine dye and flow cytometry. Western blot was used to detect cleaved PKCδ and Sytox fluorescence was used to measure caspase-3 enzyme activity.  

Exposure to 10 and 30 μM of H₂O₂ resulted in 34 and 58% increases in ROS production, respectively, compared to untreated N27 cells. Exposure to 3, 10, and 30 μM hydrogen peroxide resulted in 2-, 10-, and 9-fold increases in caspase-3 enzyme activity. Lastly, exposure to 10 and 30 μM of H₂O₂ dose-dependently induced proteolytic cleavage of PKCδ. 

Chen et al., 2009 

In vitro. PC12 and SH-SY5Y human cells were incubated with H₂O₂. The production of ROS was measured by detecting the fluorescent intensity of oxidant-sensitive probe CM-H2DCFDA. Western blot analysis was used to assess activation of MAPKs. 

Treatment with H₂O₂ for 24 h resulted in a concentration-dependent increase of ROS production at the concentrations of 0–1 mM in PC12 and SH-SY5Y cells. In comparison with PC12, SH-SY5Y cells appeared to be more sensitive to H₂O₂, thereby showing a decreased ROS production at 2 mM. Additionally, treatment of PC12 cells with H₂O₂ for 2 h increased phosphorylation of Erk1/2 and p38 in a concentration-dependent manner. Noticeably, H₂O₂-activation of JNK resulted in a robust (5–10-fold) increase of protein expression and phosphorylation of c-Jun at 0.3–1 mM. Similar results were also seen in SH-SY5Y cells (data not shown). 

Reference 

Experiment Description 

Result 

Azimzadeh et al., 2017 

In vitro. HCAECs were irradiated with 0.5 Gy of X-rays (0.5 Gy/min). Protein carbonylation and GSTO1 antioxidant level were measured with a carbonylation assay and immunoblotting, respectively. Proteins from various signaling pathways including RhoGDI, p16 and p21 were measured with immunoblotting. Measurements were taken at 1, 7, and 14 days after irradiation. 

After 7 and 14 days, carbonyl content increased 1.2-fold (insignificant increase at 1 day post-irradiation). After 1-14 days, GSTO1 decreased 0.78-fold (significant decreases at all timepoints). After 1 and 7 days, p-RhoGDI decreased 0.7-fold (non-significant decrease at 14 days post-irradiation). p16 increased 1.2-fold after 7 days and 1.5-fold after 14 days (non-significant increase at 1 day post-irradiation). p21 increased 1.2-fold after 7 and 14 days (insignificant increase at 1 day post-irradiation). 

Wortel et al., 2019 

In vitro. BAECs were irradiated with 10 Gy ¹³⁷Cs gamma rays at a rate of 1.66 Gy/min. Superoxide levels were quantified by luminescence after treatment with Diogenes Complete Enhancer Solution. The activation of the ASMase enzyme and the levels of ceramide were quantified by radioenzymatic assay to deter mine the changes on the ASMase/ceramide pathway. 

Superoxide increased by over 350% at 2 minutes post-irradiation. ASMase activity increased to a maximum 5.6-fold at 5 min post-irradiation. Ceramide increased from -500 to over 3000 pmol/106 cells at 5 minutes post-irradiation. The significance of these changes was not indicated against a control. 

Kook et al., 2015 

In vitro. MC3T3-E1 osteoblast-like cells were irradiated with X-rays (1.5 Gy/min). ROS were measured with a fluorescent probe, and SOD, CAT, and GSH antioxidant activities were determined with assay kits. Protein levels in the Nrf2/HO-1 signaling pathway were determined by either western blot or RT-PCR. 

After 1 day and 8 Gy, ROS increased 1.4-fold, GSH decreased 0.5-fold, and SOD decreased 0.5-fold. CAT was also decreased but not significantly. After 1 day and 8 Gy, Nrf2 increased 2.3-fold. After 2 days and 8 Gy, HO-1 increased 4.9-fold. After 3 days and 8 Gy, Runx2 mRNA was decreased 0.5-fold. 

Suman et al., 2013 

In vivo. Female adult C57BL/6J mice were irradiated with 1.6 Gy of ⁵⁶Fe or 2 Gy of 137Cs gamma irradiation at 1 Gy/min, then measurements were taken from the cerebral cortex until up to 12 months. ROS levels were determined with flow cytometry and 4-HNE levels were determined with immunohistochemical staining. p21 and p53 levels were determined with immunoblotting. 

All changes after ⁵⁶Fe radiation were found after both 2 and 12 months post-irradiation. Most endpoints were also increased at both time points following gamma irradiation, however, p21 only increased at 12 months by 3-fold, but not 2 months, while oxidative stress was shown at 2 months (0.2-fold increase). 

Xu et al., 2019 

In vitro. Adult male C57BL/6 mice experienced chronic cold stress for various lengths (1, 2 and 3 weeks). Brain tissue was then collected, and Western blot was used to measure MDA and proteins of MAPK (JNK, ERK and p38). 

MDA levels increased in a time-dependent manner. At 1 week, there was an approximate 3-fold increase, at 2 weeks was an approx. 4-fold increase and for 3 weeks, there was an approx. 5-fold increase in response to cold stress. Phosphorylated JNK increased by ∼10% (1 week) and ∼30% at 2 and 3 weeks compared to room temperature control. Phosphorylated ERK increased by ∼60% at 1 week, ∼150% at 2 weeks and ∼140% at 3 weeks. Phosphorylated p38 increased by ∼50% at 1 week, ∼100% at 2 weeks and ∼150% at 3 weeks. 

Chen et al., 2009 

In vitro. PC12 and SH-SY5Y human cells were incubated with hydrogen peroxide. The production of ROS was measured by detecting the fluorescent intensity of oxidant-sensitive probe CM-H2DCFDA. Western blot analysis was used to assess activation of MAPKs. 

They observed that H₂O₂ induced phosphorylation of MAPKs in a time-dependent fashion. Within 5–15 min, H₂O₂ increased phosphorylation of Erk1/2, JNK and p38, and such phosphorylation was sustained for over 2 h. Consistently, high levels of c-Jun and phospho-c-Jun were induced.