Responses of type II pneumocyte antioxidant enzymes to normoxic and hyperoxic culture

Summary Cultured type II pneumocyte responses to in vitro normoxia (95% air: 5% CO₂) or hyperoxia (95% O₂:5% CO₂) were quantified. Normoxic culture (0 to 96 h) of rabbit type II cells resulted in enhanced cell-monolayer protein and DNA content. During this same time, cellular activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH Px) decreased. Compared to cultures maintained in normoxia, hyperoxic exposure of cultures resulted in decreased cell-associated protein and DNA content. Exposure to hyperoxia also resulted in cytotoxicity as demonstrated by elevated cellular release of DNA, lactate dehydrogenase (LDH), and preincorporated 8-[¹⁴C]adenine. Cellular catalase and GSH Px activities in hyperoxic cells decreased similarly to normoxic controls. In contrast, cellular SOD activity in hyperoxic cells decreased less than in normoxic cultures. Cellular SOD activity in hyperoxic cultures, when normalized for cellular protein, but not DNA, was greater than normoxic values after 24 to 96 h of exposure. Unlike the decrease in cellular antioxidant enzymes during normoxic and hyperoxic culture, cellular LDH activity increased during both these exposures. Cellular LDH activity in 24 to 96 h hyperoxia-exposed cells increased to a lesser extent than normoxic controls. The extent of depression in LDH activity was dependent on whether the activity was normalized for cellular protein or DNA. Type II pneumocytes, which normally undergo hyperplasia and hypertrophy during hyperoxia in vivo, exhibited oxygen sensitivity in vitro. Exposure of type II cells to hyperoxia in vitro resulted in alterations in cellular SOD and LDH activities, but recognition of such changes were dependent on whether enzymatic activities were normalized for cellular DNA or protein. This work was supported by a grant from the Health Effects Institute, grant HL40458 from the National Institutes of Health, Bethesda, MD, and a grant from the American Lung Association, New York, NY.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron

In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O(2)) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cp450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H(2)O(2).     

Redox Proteomics Changes in the Fungal Pathogen Trichosporon asahii on Arsenic Exposure: Identification of Protein Responses to Metal-Induced Oxidative Stress in an Environmentally-Sampled Isolate

Trichosporon asahii is a yeast pathogen implicated in opportunistic infections. Cultures of an isolate collected from industrial wastewater were exposed for 2 days to 100 mg/L sodium arsenite (NaAsO₂) and cadmium (CdCl₂). Both metals reduced glutathione transferase (GST) activity but had no effect on superoxide dismutase or catalase. NaAsO₂ exposure increased glutathione reductase activity while CdCl₂ had no effect. Protein thiols were labeled with 5-iodoacetamido fluorescein followed by one dimensional electrophoresis which revealed extensive protein thiol oxidation in response to CdCl₂ treatment but thiol reduction in response to NaAsO₂. Two dimensional electrophoresis analyses showed that the intensity of some protein spots was enhanced on treatment as judged by SameSpots image analysis software. In addition, some spots showed decreased IAF fluorescence suggesting thiol oxidation. Selected spots were excised and tryptic digested for identification by MALDI-TOF/TOF MS. Twenty unique T. asahii proteins were identified of which the following proteins were up-regulated in response to NaAsO₂: 3-isopropylmalate dehydrogenase, phospholipase B, alanine-glyoxylate aminotransferase, ATP synthase alpha chain, 20S proteasome beta-type subunit Pre3p and the hypothetical proteins A1Q1_08001, A1Q2_03020, A1Q1_06950, A1Q1_06913. In addition, the following showed decreased thiol-associated fluorescence consistent with thiol oxidation; aconitase; aldehyde reductase I; phosphoglycerate kinase; translation elongation factor 2; heat shock protein 70 and hypothetical protein A1Q2_04745. Some proteins showed both increase in abundance coupled with decrease in IAF fluorescence; 3-hydroxyisobutyryl- CoA hydrolase; homoserine dehydrogenase Hom6 and hypothetical proteins A1Q2_03020 and A1Q1_00754. Targets implicated in redox response included 10 unique metabolic enzymes, heat shock proteins, a component of the 20S proteasome and translation elongation factor 2. These data suggest extensive proteomic alterations in response to metal-induced oxidative stress in T. asahii. Amino acid metabolism, protein folding and degradation are principally affected.     

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used α-tocopherol transfer protein knockout (α-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.     

Effect of cadmium on 24-hour pattern in expression of redox enzyme and clock genes in rat medial basal hypothalamus

The effect of cadmium (Cd) in the brain has been attributed to an increase in reactive oxygen species in cells, particularly when high amounts of the metal are given. In this study we examined the effect of a low dose of Cd (7.5 μg/day) on 24-h changes in expression of redox pathway enzyme and circadian genes in rat medial basal hypothalamus (MBH). Rats receiving CdCl₂ (5 ppm in drinking water) or tap water for 1 month were killed at six different time intervals throughout a 24 h cycle. MBH mRNA levels were measured by real-time PCR analysis. In CdCl₂ treated rats a disruption of 24-h pattern of hypothalamic gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase (HO)-1 and -2, Mn- superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase was detectable. Mean levels of MBH mRNA for HO-2, Mn-SOD and catalase augmented after Cd intake, whereas those of NOS-2 decreased. After CdCl₂ intake rats the 24-h pattern of clock gene expression in MBH seen in controls was significantly suppressed (Bmal1) or changed in phase (Per1, Per2, Cry2) while in the case of Clock significant 24-h variations were induced. The results are compatible with the view that a low amount of Cd given in tap water brought about significant changes in circadian expression of redox enzyme and clock genes in rat MBH.     

Detrimental cerebrometabolic effects of hyperoxia in newborn rats

To investigate the pathogenesis of oxygen toxicity in the newborn brain, we exposed one-day-old Sprague-Dawley albino rats to 100% O₂ and measured whole-brain high-energy phosphates, glucose, lactate, and free fatty acids (FFA) after 0, 15, 30, 60 and 120 min. Whole-brain adenosine triphosphate and creatine phosphate fell significantly from about 4.5 to 2.5 μmol-mg⁻¹ protein. Brain lactate remained at about 0.3 μmol·mg⁻¹ protein in hyperoxic rats, but increased in normoxic rats, from 0.3 to 1.3 μmol·mg⁻¹ protein at 120 min. Total FFA decreased from 30 to 15 nmol·mg⁻¹ protein during normoxia, but increased to 40 nmol·mg⁻¹ protein during hyperoxia. Undetectable in normoxic rats, arachidonic acid increased to between 4 and 6 nmol·mg⁻¹ protein during hyperoxia while oleic acid increased by two to threefold. In normoxia, palmitate decreased by 70% from 12 to 4 nmol·mg⁻¹ protein whereas in hyperoxia it remained at 10 nmol·mg⁻¹ protein. Normobaric 100% O₂ has detrimental metabolic effects on the neonatal brain which cannot be attributed to cerebral vasospasm or seizure-induced cerebral anoxia because lactic acidosis was not observed. FFA changes suggest that a likely explanation is membrane lipid peroxidation from O₂-induced free radicals.     

Neuroglobin Involvement in the Course of Arsenic Toxicity in Rat Cerebellar Granule Neurons

Exposure to arsenic in drinking water results in a widespread environmental problem in the world, and the brain is a major target. Neuroglobin is a vertebrate heme protein regarded as playing neuroprotective role in hypoxia or oxidative stress. In this study, we investigated the toxic effects of sodium arsenite (NaAsO₂) on primary cultured rat cerebellar granule neurons (CGNs) and detected neuroglobin (Ngb) expression in rat CGNs exposed to NaAsO₂. Our results show that apoptosis was obviously induced by NaAsO₂ treatment in rat CGNs by annexin V-fluorescein isothiocyanate assay. Intracellular reactive oxygen species generation increased significantly in the cells exposed to NaAsO₂, and the apoptotic effects could be partially reversed by antioxidant N-acetyl-l-cysteine. Ngb protein and mRNA expression were significantly downregulated in rat CGNs shortly after NaAsO₂ exposure and then upregulated after a longer time of exposure. Furthermore, mRNA expression changed more than protein expression and the toxic effect of NaAsO₂ on Ngb expression is dose dependent. Higher Ngb expression was also detected in rat cerebellum, but not in other parts (cerebrum, hippocampus, and midbrain) of the brain exposed to NaAsO₂ for 16 weeks. Taken together, cytotoxic effects of NaAsO₂ on rat CGNs is induced at least partly by oxidative stress and Ngb may influence the course of arsenic toxicity in rat CGNs and rat cerebellum.     

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O₂ ^·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd²⁺ treatments and the expression of the NADPH oxidase putative gene related to O₂ ^·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl₂ and almost completely depleted by 0.5 mM CdCl₂.     

Cadmium stress in sugar cane callus cultures: Effect on antioxidant enzymes

Catalase (CAT) and superoxide dismutase (SOD) are antioxidant enzymes which are important in the metabolism of reactive oxygen species (ROS), and can be induced by environmental stresses including cadmium (Cd), a heavy metal toxic to living organisms. Sugar cane (Saccharum officinarumL.) in vitro callus cultures were exposed to CdCl₂ and the activities of CAT and SOD were analysed. Lower concentrations of CdCl₂, such as 0.01 and 0.1 mM caused a significant increase in callus growth, whereas 0.5 and 1 mM CdCl₂ strongly inhibited growth of the callus cultures, but only after 9 days of CdCl₂ treatment. Red-brown patches were also observed in calluses exposed to 0.5 and 1 mM CdCl₂. Calluses grown in 0.01 and 0.1 mM CdCl₂ did not exhibit any changes in CAT activity even after 15 days of growth in the presence of CdCl₂. However, for calluses grown in higher concentrations of CdCl₂ (0.5 and 1 mM), a rapid increase in CAT activity was detected, which was 14-fold after 15 days. Furthermore, up to five CAT isoforms were observed in callus tissue. Total SOD activity did not exhibit any major variation. One Mn-SOD and two Cu/Zn-SOD isoenzymes were observed in callus cultures and none exhibited any variation in response to the CdCl₂ treatments. The results suggested that in sugar cane callus cultures, CAT may be the main antioxidant enzyme metabolizing H₂O₂.     

N-acetyl cysteine reduces oxidative toxicity,apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a common inflammatory and oxidant disease with an uncertain pathogenesis. N-acetyl cysteine (NAC) decreases oxidative stress, intracellular free calcium ion [Ca²⁺]_i, and apoptosis levels in human neutrophil. We aimed to investigate the effects of NAC on apoptosis, oxidative stress, and Ca²⁺ entry through transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 2 (TRPM2) channels in neutrophils from patients with PCOS. Neutrophils isolated from PCOS group were investigated in three settings: (1) after incubation with TRPV1 channel blocker capsazepine or TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB), (2) after supplementation with NAC (for 6 weeks), and (3) with combination (capsazepine + 2-APB + NAC) exposure. The neutrophils in TRPM2 and TRPV1 experiments were stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM) and capsaicin (10 μM) as concentration agonists, respectively. Neutrophil lipid peroxidation and capsaicin-induced increase in [Ca²⁺]_i concentrations were reduced by capsazepine and NAC treatments. However, the [Ca²⁺]_i concentration did not change by fMLP stimulation. Neutrophil lipid peroxidation, apoptosis, caspase-3, caspase-9, cytosolic reactive oxygen species production, and mitochondrial membrane depolarization values were decreased by NAC treatment although neutrophil glutathione peroxidase and reduced glutathione levels were increased by the NAC treatment. Serum lipid peroxidation, luteinizing hormone, testosterone, insulin, interleukin-1 beta, and homocysteine levels were decreased by NAC treatment although serum vitamin A, beta-carotene, vitamin E, and total antioxidant status were increased by the NAC treatment. In conclusion, NAC reduced oxidative stress, apoptosis, cytokine levels, and Ca²⁺ entry through TRPV1 channel, which provide supportive evidence that oxidative stress and TRPV1 channel plays a key role in etiology of PCOS.     

Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC)

The antioxidant potential of N-acetylcysteine amide (NACA), also known as AD4, was assessed by employing different in vitro assays. These included reducing power, free radical scavenging capacities, peroxidation inhibiting activity through linoleic acid emulsion system and metal chelating capacity, as compared to NAC and three widely used antioxidants, α-tocopherol, ascorbic acid and butylated hydroxytoluene (BHT). Of the antioxidant properties that were investigated, NACA was shown to possess higher 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging ability and reducing power than NAC, at all the concentrations, whereas the scavenging ability of H₂O₂ differed with concentration. While NACA had greater H₂O₂ scavenging capacity at the highest concentration, NAC was better than NACA at lower concentrations. NAC and NACA had a 60% and 55% higher ability to prevent β-carotene bleaching, respectively, as compared to control. The chelating activity of NACA was more than 50% that of the metal chelating capacity of EDTA and four and nine times that of BHT and α-tocopherol, respectively. When compared to NACA and NAC; α-tocopherol had higher DPPH scavenging abilities and BHT and α-tocopherol had better β-carotene bleaching power. These findings provide evidence that the novel antioxidant, NACA, has indeed enhanced the antioxidant properties of NAC.     

Postnatal Hyperoxia Exposure Differentially Affects Hepatocytes and Liver Haemopoietic Cells in Newborn Rats

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O₂ (moderate hyperoxia), or 95% O₂ (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.     

NF-κB involvement in hyperoxia-induced myocardial damage in newborn rat hearts

Premature newborns are frequently exposed to hyperoxia ventilation and some literature data indicate the possibility of hyperoxia-induced myocardial damage. Since nuclear factor κB (NF-κB) is a crucial signaling molecule involved in physiological response to hyperoxia in different cell types as well as in various tissues, our attention has been focused on the role played by NF-κB pathway in response to moderate and severe hyperoxia exposure in rat neonatal heart tissue. Akt and IκBα levels, involved in NF-κB activation, along with the balance between apoptotic and survival pathways have also been investigated. Experimental design of the study has involved exposure of newborn rats to room air (controls), 60 % O₂ (moderate hyperoxia), or 95 % O₂ (severe hyperoxia) for the first two postnatal weeks. Morphological analysis shows a less compact tissue in rat heart exposed to moderate hyperoxia and a decreased number of nuclei in samples exposed to severe hyperoxia. A significant increase of NF-κB positive nuclei percentage and p-IκBα expression in samples exposed to 95 % hyperoxia compared to control and to 60 % hyperoxia is evidenced; in parallel, an increase of pAkt/Akt ratio in both samples exposed to 95 and 60 % hyperoxia is shown. Furthermore, a more evident cytochrome c/Apaf-1 immunocomplex and a decreased Bcl2 expression in 95 % hyperoxia-exposed sample compared to 60 % exposed one is evidenced. In conclusion, our findings suggest the involvement of the NF-κB pathway and Akt signaling in the mechanisms of myocardial hyperoxic damage in the newborns, with particular reference to the induction of oxidative stress-related apoptosis.     

Proinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury

BackgroundHyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O₂) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response.MethodsWild-type and iNOS-deficient mice were exposed to normoxia, 60% O₂ or >95% O₂ for 72 h.ResultsExposure to >95% O₂ resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice.ConclusionTaken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury.