Carbon monoxide and bilirubin from heme oxygenase-1 suppresses reactive oxygen species generation and plasminogen activator inhibitor-1 induction

Heme oxygenase-1 (HO-1) responds to a variety of oxidative stresses. We examined whether HO-1 expression influences pro-thrombotic processes, in which the involvement of oxidative stress has been reported. Since HO-1 knockout mice with a C57/BL6J background were not viable, embryonic cells from HO-1 deficient mice (E11.5) were used. Cell viability, the level of plasminogen activator inhibitor-1 (PAI-1) expression and reactive oxygen species (ROS) generation of HO-1 deficient cells in response to the exposures to hydrogen peroxide and oxidized LDL were compared to those with wild-type cells. We also examined the effects of glutathione (GSH), desferrioxamine (DFO) and diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) as well as of the HO reaction products, bilirubin (BR) and carbon monoxide (CO) on PAI-1 expression and ROS generation. PAI-1 expression and ROS generation were markedly elevated in HO-1 deficient cells compared to wild-type cells. Exposure to oxidized LDL significantly elevated PAI-1 expression and ROS production in HO-1 deficient cells. Interestingly, these increases in HO-1 deficient cells were significantly lowered by BR, CO, GSH and DPI while DFO had little effect. Furthermore, BR and CO were effective to improve viabilities of HO-1 deficient cells. These results suggest that HO-1 may be required to suppress ROS generation and the production of pro-thrombotic molecules such as PAI-1.     

The effects of dopamine on antioxidant enzymes activities and reactive oxygen species levels in soybean roots

In the current work, we investigated the effects of dopamine, an neurotransmitter found in several plant species on antioxidant enzyme activities and ROS in soybean (Glycine max L. Merrill) roots. The effects of dopamine on SOD, CAT and POD activities, as well as H₂O₂, O₂^•−, melanin contents and lipid peroxidation were evaluated. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.1 to 1.0 mM dopamine, in a growth chamber (25°C, 12 h photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 h. Significant increases in melanin content were observed. The levels of ROS and lipid peroxidation decreased at all concentrations of dopamine tested. The SOD activity increased significantly under the action of dopamine, while CT activity was inhibited and POD activity was unaffected. The results suggest a close relationship between a possible antioxidant activity of dopamine and melanin and activation of SOD, reducing the levels of ROS and damage on membranes of soybean roots.     

Nicotinic receptor activation by epibatidine induces heme oxygenase-1 and protects chromaffin cells against oxidative stress

Activation of neuronal nicotinic acetylcholine receptors (nAChR) provides neuroprotection against different toxic stimuli that often lead to overproduction of reactive oxygen species (ROS) and cell death. ROS production has been related with disease progression in several neurodegenerative pathologies such as Alzheimer's or Parkinson's diseases. In this context, we investigated here if the exposure of bovine chromaffin cells to the potent nAChR agonist epibatidine protected against rotenone (30 micromol/L) plus oligomycin (10 micromol/L) (rot/oligo) toxicity, an in vitro model of mitochondrial ROS production. Epibatidine induced a concentration- and time-dependent protection, which was maximal at 3 mumol/L after 24 h. Pre-incubation with dantrolene (100 micromol/L) (a blocker of the ryanodine receptor channel), chelerythrine (1 micromol/L) (a protein kinase C inhibitor), or PD98059 (50 micromol/L) (a MEK inhibitor), aborted epibatidine-elicited cytoprotection. Mitochondrial depolarization, ROS, and caspase 3 active produced by rot/oligo were also prevented by epibatidine. Epibatidine doubled the amount of heme oxygenase-1 (HO-1), a critical cell defence enzyme against oxidative stress. Furthermore, the HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride reversed the epibatidine protecting effects and HO-1 inducer Co (III) protoporphyrin IX dichloride exhibited neuroprotective effects by itself. The results of this study point to HO-1 as the cytoprotective target of nAChR activation through the following pathway: endoplasmic reticulum Ca(2+)-induced Ca(2+)-release activates the protein kinase C/extracellular regulated kinase/HO-1 axis to mitigate mitochondrial depolarization and ROS production. This study provides a mechanistic insight on how nAChR activation translates into an antioxidant and antiapoptotic signal through up-regulation of HO-1.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3-8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9-3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3–8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9–3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean

Our previous investigation reported the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean. In this study, soybean seeds treated with static magnetic fields of 150 and 200 mT for 1 h were evaluated for reactive oxygen species (ROS) and activity of antioxidant enzymes. Superoxide and hydroxyl radicals were measured in embryos and hypocotyls of germinating seeds by electron paramagnetic resonance spectroscopy and kinetics of superoxide production; hydrogen peroxide and antioxidant activities were estimated spectrophotometrically. Magnetic field treatment resulted in enhanced production of ROS mediated by cell wall peroxidase while ascorbic acid content, superoxide dismutase and ascorbate peroxidase activity decreased in the hypocotyl of germinating seeds. An increase in the cytosolic peroxidase activity indicated that this antioxidant enzyme had a vital role in scavenging the increased H(2)O(2) produced in seedlings from the magnetically treated seeds. Hence, these studies contribute to our first report on the biochemical basis of enhanced germination and seedling growth in magnetically treated seeds of soybean in relation to increased production of ROS.     

Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition

Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.     

Sodium meta-arsenite induced reactive oxygen species in human red blood cells: impaired antioxidant and membrane redox systems,haemoglobin oxidation,and morphological changes

Arsenic (As) is an air and water toxicant that causes cancer in multiple organs. Humans are exposed to As through contaminated water. We have examined the cytotoxicity of sodium meta-arsenite (SA), an As(III) compound, in human red blood cells (RBC) under in vitro conditions. Haemolysates were prepared from human RBC treated with different concentrations of SA (0.1–5.0?mM) for 5?h at 37?°C. SA treatment of RBC caused significant increase in methaemoglobin formation, protein and lipid oxidation, and nitric oxide levels. It also resulted in decrease in glutathione levels, methaemoglobin reductase activity and plasma membrane redox system. SA exposure also inhibited the pathways of glucose metabolism while increasing AMP deaminase and glyoxalase-I. It impaired the enzymatic and non-enzymatic antioxidant defence systems which resulted in decreased antioxidant power and a compromised ability to quench free radicals. SA exposure also damaged the membrane since it decreased the activity of membrane bound enzymes, increased the osmotic fragility of treated cells and induced gross morphological changes. This cytotoxicity was the result of oxidative damage since the production of reactive oxygen species (ROS) was increased in SA treated erythrocytes. Thus As(III) causes extensive damage to RBC which impairs their antioxidant system and alters the major cellular metabolic pathways. All this has the potential to lower the oxygen carrying capacity of RBC and reduce their lifespan in blood.     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.     

Analysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p<0.05 or p<0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.     

Heme oxygenase-1 protects bone marrow mesenchymal stem cells from iron overload through decreasing reactive oxygen species and promoting IL-10 generation

Iron overload (IO) caused by frequent blood transfusion in hematological diseases has become a major concern. In this study, up-regulation of heme oxygenase-1 (HO-1), a protector against oxidative stress, was observed in bone marrow mesenchymal stem cells (BMMSCs) at the early stage of IO and had favorable prognosis in an IO mouse model. Given that the protective role of HO-1 in IO damage of BMMSCs was still unknown, the mechanism was explored in vitro and in vivo. BMMSCs were transfected with HO-1/siHO-1 in vitro, and the mouse model was established to further evaluate the effect of HO-1 on IO in vivo. As a result, HO-1 decreased the apoptotic rate of BMMSCs with IO through reducing intracellular reactive oxygen species (ROS) but increasing IL-10 secretion. In addition, IL-10 was mediated by HO-1 via the ERK pathway. Intracellular iron was down-regulated by hepcidin depending on IL-10. In conclusion, HO-1 protects BMMSCs from ROS by secreting IL-10 upon iron overload.     

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l -cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.     

HO-1在U2OS细胞DOX耐药中的作用及分子机制研究

目的:探讨血红素氧合酶-1(HO-1)在骨肉瘤U2OS细胞多柔比星(DOX)耐药中的作用及相关分子机制。方法:体外培养U2OS细胞,建立U2OS-DOX耐药株,分为U2OS-WT组和U2OS-DOX组。采用siRNA HO-1转染U2OS-DOX细胞,CCK-8法检测细胞活性;RT-PCR法检测缺氧诱导因子1(HIF-1α)及HO-1的mRNA表达;WB法检测HIF-1α及HO-1的蛋白表达水平;流式细胞仪检测罗丹明Rh123在细胞内的蓄积。结果:DOX可降低U2OS细胞活性并随剂量的增加愈加明显,这种诱导作用可以被抗氧化剂(NAC)所逆转(P<0.01)。U2OS-DOX组HIF-1α及HO-1的mRNA和蛋白表达以及P糖蛋白(P-gp)表达水平均显著增加(P<0.05)。转染可恢复U2OS-DOX细胞对DOX化疗敏感性并增加其对Rh123的蓄积(P<0.001)。结论:HO-1可能通过抗氧化应激、增加化疗药物的蓄积等机制发挥U2OS细胞对DOX的耐药性。     

拟南芥CEO1基因在氯化镉诱导的氧化胁迫中的作用

以拟南芥ceo1、突变体为材料,研究CEO1（clone eight-one）在镉胁迫条件下作用的结果表明,与野生型植株相比,150μmol·L^-1的CdCl2处理10d后,拟南芥ceo1突变体表现为植株生长矮小,叶片卷曲发黄,根系短小。镉处理后,拟南芥突变体幼苗叶中H2O2的积累较多;镉处理1h后的突变体中抗坏血酸过氧化物酶（APX）活性明显上升,至2h时又开始下降,而镉处理2h后,野生型APX活性才开始增加。镉处理2h后的野生型的谷胱甘肽还原酶（GR）显著增加,而突变体无明显变化。两种类型拟南芥的超氧化物歧化酶（SOD）与过氧化氢酶（CAT）的活性没有明显差异。     

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18?days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.     

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.