Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5 h following LPS treatment (100 μg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10 μM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.     

锰作用下PC12细胞的氧化应激机制研究

目的：以鼠嗜铬神经瘤细胞（PC12）为模型,筛选锰对神经细胞增殖抑制作用的时间及剂量,观察锰作用下PC12细胞的氧化应激反应与细胞形态学、生化指标改变和丝裂原活化蛋白激酶pp38（p38MAPKs）的活化表达。方法：用200,400,600,800μmol/LMnCl2的培养液,分别作用对数生长期PC12细胞1,2,3,4d后,用MTT筛选锰的细胞毒性剂量;测定200-600μmol/L MnCl2作用4d后,PC12细胞还原型谷胱甘肽和丙二醛含量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PC12细胞基因组DNA的影响。western-blot法检测p-p38。结果：MTT实验显示200~800μmol/LMnCl2作用1,2,3,4d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/LMnCl2作用4d对PC12的抑制率可达50%以上。200-600μmol/LMnCl2作用于细胞4d后,随着浓度的升高,还原型GSH逐渐降低,MDA的含量逐渐升高;600μmol/LMnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化。Western-blot实验显示600μmol/LMnCl2作用1,2,3,4dp-p38逐渐升高,3d时较对照组增加6.6倍（n=3,p〈0.05）,200,400,600μmol/L MnCl2作用4d时,磷酸化蛋白38（p-p38）也逐渐升高,400μmol/L MnCl2作用4d时较对照组升高了4.7倍（n=3,p〈0.05）。结论：PC12细胞在锰作用下发生氧化应激反应,上调p-p38,诱导细胞凋亡,细胞增殖抑制。     

Oxidative stress causes a general, calcium-dependent degradation of mitochondrial polynucleotides

Oxidative stress has many effects on biological cells, including the modulation of gene expression. Reactive oxygen species are known to up-regulate and down-regulate RNA expression in prokaryotic and eukaryotic cells. We have previously reported that a preferential and calcium-dependent down-regulation of mitochondrial RNAs occurs when HA-1 hamster fibroblasts are exposed to hydrogen peroxide. Here we extend these studies to determine whether this down-regulation is specific to mitochondria RNA or involves general polynucleotide degradation. Degradation and associated decreases in the levels of 16S mitochondrial rRNA following exposure of cells to 400 μM hydrogen peroxide were found to be dependent on calcium at 2 and 5 h. Degradation of mitochondrial genomic DNA was also observed following peroxide exposure, and occurred at similar time points as for mitochondrial RNA degradation. As with mitochondrial RNA degradation, this mitochondrial genomic DNA degradation was dependent on calcium. These results indicate that there is a general, calcium-dependent degradation of mitochondrial polynucleotides following exposure of HA-1 fibroblasts to oxidative stress, and suggest that a dramatic shut-down in mitochondrial biosynthesis is an early-stage response to oxidative stress.     

Oxidative and nitrative stress markers in glaucoma

Glaucoma is a progressive optic neuropathy and is the leading cause of blindness in the United States and other industrialized countries. Elevated pressure in the eye is a risk factor for glaucoma and indeed experimental studies of induced pressure elevation in nonhuman primate's results in typical glaucomatous optic nerve damage. However, normal intraocular pressure can also lead to loss of vision in glaucoma. Although the initiating causes leading to glaucoma are unknown, oxidative and nitrative stress appears to play a role in the progressive neuronal death that is characteristic of glaucomatous optic nerve damage. Increased markers of oxidative stress that have been reported in glaucoma include protein nitrotyrosine, carbonyls in proteins, lipid oxidation products and oxidized DNA bases. Studies have also highlighted the role of nitric oxide in glaucoma by reporting the presence of inducible nitric oxide synthase in the iris-ciliary body, retina and in the glaucomatous optic nerve head of experimental rat models. This review discusses the role of reactive oxygen and nitrogen species in the pathogenesis of glaucoma and examines the relevance of antioxidants in neurodegeneration associated with the disease. It is concluded that oxidative and nitrative stress have a pathogenic role in glaucoma.     

Oxidative stress impairs endocytosis of the scavenger receptor class A

We report the characterization of a cell system employing Chinese hamster ovary (CHO) cells and CHO cells transfected with the scavenger receptor class A (CHO-SRA) using extracellularly produced reactive oxygen species (ROS) in order to study the endocytic function of the scavenger receptor. The oxidative environment was produced using tert-butyl hydroperoxide (TBH) and characterized by flow cytometry and cell viability. Once an adequate oxidative environment was established, binding and internalization studies of radiolabeled acetylated LDL particles (125I-labeled Ac-LDL) with CHO-SRA cells were carried out. RT-PCR analysis using total RNAs from CHO-SRA cells revealed that oxidative stress does not alter the expression of the scavenger receptor. However, internalization of 125I-labeled Ac-LDL through this receptor carried out by these cells was completely abolished under extracellularly oxidative conditions. Together, these results support the idea that an oxidative stress produced extracellularly, inhibiting the endocytosis of the scavenger receptor, could help to understand and explain the mechanisms by which several physiologically important ligands are accumulated in the extracellular space with its consequent cell damage.     

Oxidative stress and apoptosis in preeclampsia

We aimed to determine the oxidative stress and antioxidant status in preeclamptic placenta. Also, we investigated the apoptotic index of villous trophoblast and proliferation index of cytotrophoblasts. The study included 32 pregnant with preeclampsia and 31 normotensive healthy pregnant women. Malondialdehyde (MDA) and total antioxidant status (TAS) levels were measured in the placenta. For detection of apoptosis and proliferation in trophoblast, apoptosis protease activating factor 1 (APAF-1) and Ki-67 were used. Placental MDA levels in preeclamptic women were significantly higher than normal pregnancies (p = 0.002). There was no significant difference between the groups in the TAS levels of placenta (p = 0.773). Also, the apoptotic index in villous trophoblasts increased (p < 0.001), but proliferation index did not change in preeclampsia (p = 0.850). Increased oxidative stress and apoptosis in pathological placenta are not balanced by antioxidant systems and proliferation mechanisms.     

Correlation between melanogenic and catalase activity in in vitro human melanocytes: a synergic strategy against oxidative stress

UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.     

Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse

Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and function. These processes appear in many cases to be interrelated, and this review will examine current understanding of these processes on the equine sperm function.     

Oxidative stress and antioxidant defenses in pregnant women

Abstract

Objectives

Oxidative stress (OS) is defined as an imbalance in the production of reactive oxygen species and the capacity of antioxidant defenses. The objective of this work was to investigate OS and antioxidant capacity in pregnant women.

Methods

Parameters of the oxidative status and antioxidant capacity in serum and whole blood were evaluated in thirty-nine women with normal pregnancy.

Results

The assessment of antioxidants indicated an increase in superoxide dismutase and catalase activities (P < 0.05 and P < 0.01) and a decrease in ascorbic acid levels and the total content of sulfhydryl (P < 0.05 and P < 0.001). Additionally, when the pro-oxidant system was investigated we found an increase (P < 0.01) in malondialdehyde and no significant change (P > 0.05) in protein carbonylation.

Discussion

This study demonstrates that there is a change in the pro-oxidant and antioxidant defenses associated with body and circulation changes that are inherent to the pregnancy process.     

Oxidative stress as a cofactor in spinocerebellar ataxia type 2

Abstract

Spinocerebellar ataxia type 2 (SCA2) is a redox-sensitive neurodegenerative disease affecting the cerebellum, fibre connections in the cerebellum, the peripheral nervous system, and extracerebellar central pathways. Currently, Cuba has the highest reported global rate for this disease. The aim of this review article is to summarize and discuss the current knowledge about evidence of oxidative stress during SCA2. Recent reports have suggested that ataxin 2 and other related factors contribute to the redox imbalance in this disease. It is important to recognize and clarify the molecular mechanisms associated with the redox imbalance to consider ataxias innovative approaches to counteract oxidative stress-induced tissue damage, through alternative therapeutic or nutritional intervention in SCA2 and related diseases.     

Oxidative balance score and oxidative stress biomarkers in a study of Whites,African Americans,and African immigrants

Abstract

Context: Oxidative balance score (OBS) is a composite measure of multiple pro- and antioxidant exposures.

Objective: To investigate associations of OBS with F2-isoprostanes (FIP), mitochondrial DNA copy number (mtDNA), and fluorescent oxidative products (FOP), and assess inter-relationships among the biomarkers.

Methods: In a cross-sectional study, associations of a thirteen-component OBS with biomarker levels were assessed using multivariable regression models.

Results: Association of OBS with FIP, but not with FOP, was in the hypothesized direction. The results for mtDNA were unstable and analysis-dependent. The three biomarkers were not inter-correlated.

Conclusions: Different biomarkers of oxidative stress may reflect different biological processes.     

Oxidative stress and ischemia-modified albumin in chronic ischemic heart failure

Abstract

Objectives

Knowledge about the role of oxidative stress in human diseases, including cardiovascular system disorders, emphasizes the need for reliable markers of oxidative stress. Here, we evaluated the levels of the novel marker ischemia-modified albumin (IMA), albumin-adjusted IMA (adj-IMA), and the IMA/serum albumin ratio (IMAR) in patients with chronic ischemic heart failure (CIHF).

Methods

A total of 55 patients with CIHF and 40 age- and sex-matched healthy individuals were included in the study. Serum levels of IMA, total antioxidant status, and total oxidant status were analyzed, and the adj-IMA level, IMAR, and oxidative stress index were calculated.

Results

Serum IMA, IMAR, total oxidant status levels, and oxidative stress index were significantly higher in patients with CIHF than in the controls (all P < 0.0001), whereas albumin and total antioxidant status levels were significantly lower in the CIHF patients (P < 0.0001 and P = 0.0004, respectively). However, there was no significant difference in serum adj-IMA levels between the groups (P = 0.8).

Discussion

We observed impaired oxidant/antioxidant status in favor of oxidative stress in CIHF patients. Oxidative stress may be a key factor in the development of hypoalbuminemia in CIHF. Further studies are needed to establish the relationships among IMA, albumin, and redox balance in CIHF.     

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells

Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC₂ receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A¹¹]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 μM monensin but not by 10 μg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 μM monensin and 10 μg/ml cycloheximide.     

Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents

Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.