What happens with ageing: decline or remodeling of opsonin‐independent phagocyte oxidative activity?

Changes that occur with age in the opsonin‐independent oxidative activity of peripheral phagocytes in whole blood were examined by means of luminol chemiluminescence (LCL). The chemiluminescence was registered simultaneously by non‐stimulated and stimulated cells and the age‐related alterations of total and extracellular generation of reactive oxygen species (ROS) were studied using model systems. It was found that the rate of phagocyte activation by the glass surface of the measuring chambers, assessed by the time of the peak appearance after the start of LCL response, increased. However, the maximum oxidative activity and the integral oxidative capacity of the cells during adhesion, evaluated by the maximum LCL intensity and the area under the LCL curve, respectively, declined. No age‐dependence of formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐stimulated oxidative cellular activity for total ROS generation was detected. The maximum oxidative activity and the integral oxidative capacity of peripheral phagocytes to generate extracellular superoxide in response to fMLP was decreased. The likely causes for the observed alterations in phagocyte function are discussed and an analysis of the obtained results is given on the background of the contradictory data published on phagocyte oxidative activity age‐related changes. Copyright © 2009 John Wiley & Sons, Ltd.     

Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-β) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.     

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1β, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.     

alpha-Pinene inhibits growth and induces oxidative stress in roots

BACKGROUND AND AIMS: Determining the mode of action of allelochemicals is one of the challenging aspects in allelopathic studies. Recently, allelochemicals have been proposed to cause oxidative stress in target tissue and induce an antioxidant mechanism. alpha-Pinene, one of the common monoterpenoids emitted from several aromatic plants including forest trees, is known for its growth-inhibitory activity. However, its mechanism of action remains unexplored. The aim of the present study was to determine the inhibitory effect of alpha-pinene on root growth and generation of reactive oxygen species, as indicators of oxidative stress and changes in activities of antioxidant enzymes. METHODS: Effects of alpha-pinene on early root growth were studied in five test species, Cassia occidentalis, Amaranthus viridis, Triticum aestivum, Pisum sativum and Cicer arietinum. Electrolyte leakage, lipid peroxidation, hydrogen peroxide generation, proline accumulation, and activities of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR) were studied in roots of C. occidentalis. KEY RESULTS: alpha-Pinene inhibited the radicle growth of all the test species. Exposure of C. occidentalis roots to alpha-pinene enhanced solute leakage, and increased levels of malondialdehyde, proline and hydrogen peroxide, indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes SOD, CAT, GPX, APX and GR were significantly elevated, thereby indicating the enhanced generation of reactive oxygen species (ROS) upon alpha-pinene exposure. Increased levels of scavenging enzymes indicates their induction as a secondary defence mechanism in response to alpha-pinene. CONCLUSIONS: It is concluded that alpha-pinene inhibits early root growth and causes oxidative damage in root tissue through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and elevated antioxidant enzyme levels.     

Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca²⁺ influx suggest that a rise in cytosolic Ca²⁺ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca²⁺ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.     

Reactive oxygen species and ischemic cerebrovascular disease

Stroke is an emerging major health problem often resulting in death or disability. Hyperlipidemia, high blood pressure and diabetes are well established risk factors. Endothelial dysfunction associated with these risk factors underlies pathological processes leading to atherogenesis and cerebral ischemic injury. While mechanisms of disease are complex, endothelial dysfunction involves decreased nitric oxide (NO) and elevated levels of reactive oxygen species (ROS). At physiological levels, ROS participate in regulation of cellular metabolism. However, when ROS increase to toxic levels through imbalance of production and neutralization by antioxidant enzymes, they cause cellular injury in the form of lipid peroxidation, protein oxidation and DNA damage. Central nervous system cells are more vulnerable to ROS toxicity due to their inherent higher oxidative metabolism and less antioxidant enzymes, as well as higher content of membranous fatty acids. During ischemic stroke, ROS concentration rises from normal low levels to a peak point during reperfusion possibly underlying apoptosis or cellular necrosis. Clinical trials and animal studies have shown that natural compounds can reduce oxidative stress due to excessive ROS through their antioxidant properties. With further study, we may be able to incorporate these compounds into clinical use with potential efficacy for both the treatment and prevention of stroke.     

Lipid peroxidation-mediated cytotoxicity and DNA damage in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. In the present study, we evaluated lipid peroxidation-mediated cytotoxicity and oxidative DNA damage in U937 cells. Upon exposure of U937 cells to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the cells exhibited a reduction in viability and an increase in the endogenous production of reactive oxygen species (ROS), as measured by the oxidation of 2',7'-dichlorodihydrofluorescein. In addition, a significant decrease in the intracellular GSH level and the activities of major antioxidant enzymes were observed. We also observed lipid peroxidation-mediated oxidative DNA damage, reflected by an increase in 8-OH-dG level and loss of the ability of DNA to renature. When the cells were pretreated with the antioxidant N-acetylcysteine (NAC) or the spin trap alpha-phenyl-N-t-butylnitrone (PBN), lipid peroxidation-mediated cytotoxicity in U937 cells was protected. This effect seems to be due to the ability of NAC and PBN to reduce ROS generation induced by lipid peroxidation. These results suggest that lipid peroxidation resulted in a pro-oxidant condition of U937 cells by the depletion of GSH and inactivation of antioxidant enzymes, which consequently leads to a decrease in survival and oxidative damage to DNA. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in oxidative stress-induced cellular damage.     

Oxidative stress in silicosis: Evidence for the enhanced clearance of free radicals from whole lungs

We hypothesized that reactive oxygen species (ROS) may be involved in the pathogenesis of silicosis. To investigate ROS' dependent pathophysiological processes during silicosis we studied the kinetic clearance of instilled stable nitroxide radicals (TEMPO). Antioxidant enzymes' superoxide dismutase (SOD) and glutathione peroxidase (GPx), and lipid peroxidation were also studied in whole lungs of rats exposed to crystalline silica (quartz) and sham exposed controls. Low frequency L-band electron spin resonance spectroscopy was used to measure the clearance of TEMPO in whole-rat lungs directly. The clearance of TEMPO followed first order kinetics showing significant differences in the rate for clearance between the diseased and sham exposed control lungs. Comparison of TEMPO clearance rates in the sham exposed controls and silicotic rats showed an oxidative stress in the rats exposed to quartz. Studies on the antioxidant enzymes SOD and GPx in the lungs of silicotic and sham exposed animals supported the oxidative stress and accelerated clearance of TEMPO by up regulated levels of enzymes in quartz exposed animals. Increased lipid peroxidation potential in the silicotics also supported a role for enhanced generation of ROS in the pathogenesis of silica-induced lung injury. These in vivo experiments directly demonstrate, for the first time, that silicotic lungs are in a state of oxidative stress and that increased generation of ROS is associated with enhanced levels of oxidative enzymes and lipid peroxidation. This technique offers great promise for the elucidation of ROS induced lung injury and development of therapeutic strategies for the prevention of damage.     

Influence of novel naphthalimide-based organoselenium on genotoxicity induced by an alkylating agent: the role of reactive oxygen species and selenoenzymes

Abstract

Objective

The protection conferred by a series of synthetic organoselenium compounds against genotoxicity and oxidative stress induced by a reference mutagen cyclophosphamide (CP) was assessed.

Method

Genotoxicity was induced in mice by CP treatment (25 mg/kg b.w.) for 10 consecutive days. Organoselenium compounds (3 mg/kg b.w.) were administered orally in a concomitant and pretreatment schedule. DNA damage in peripheral blood lymphocytes and frequency of chromosomal aberration in the bone marrow cells were measured. Liver tissues were collected for analysis of the activity of antioxidant and detoxifying enzymes, lipid peroxidation (LPO) level, glutathione content, and histopathology.

Results

Exposure to CP not only led to a significant increase in the percent of chromosomal aberration and DNA damage, but also enhanced generation of hepatic reactive oxygen species (ROS) and LPO level. The organoselenium compounds demonstrated marked functional protection against CP-induced genotoxicity. DNA damage and chromosomal aberration along with ROS generation were attenuated in the organoselenium-treated mice compared with the CP-treated control mice. CP caused marked depression in the activities of the selenoenzymes (glutathione peroxidase (GPx) and thioredoxin reductase (TRxR)) and other detoxifying and antioxidant enzymes, while treatment with organoselenium compounds restored all these activities towards normal.

Discussion

The protective effect of these compounds may be primarily associated with the improvement of the activity of antioxidant and detoxifying enzymes (including the selenoenzymes, GPx, and TRxR) that are known to protect the DNA and other cellular components from oxidative damage.     

Wavelength dependence of biological damage induced by UV radiation on bacteria

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.     

Systemic oxidative stress is implicated in the pathogenesis of brain edema in rats with chronic liver failure

Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE); however, a correlation between blood ammonia levels and HE severity remains controversial. It is believed oxidative stress plays a role in modulating the effects of hyperammonemia. This study aimed to determine the relationship between chronic hyperammonemia, oxidative stress, and brain edema (BE) in two rat models of HE: portacaval anastomosis (PCA) and bile-duct ligation (BDL). Ammonia and reactive oxygen species (ROS) levels, BE, oxidant and antioxidant enzyme activities, as well as lipid peroxidation were assessed both systemically and centrally in these two different animal models. Then, the effects of allopurinol (xanthine oxidase inhibitor, 100mg/kg for 10days) on ROS and BE and the temporal resolution of ammonia, ROS, and BE were evaluated only in BDL rats. Similar arterial and cerebrospinal fluid ammonia levels were found in PCA and BDL rats, both significantly higher compared to their respective sham-operated controls (p<0.05). BE was detected in BDL rats (p < 0.05) but not in PCA rats. Evidence of oxidative stress was found systemically but not centrally in BDL rats: increased levels of ROS, increased activity of xanthine oxidase (oxidant enzyme), enhanced oxidative modifications on lipids, as well as decreased antioxidant defense. In PCA rats, a preserved oxidant/antioxidant balance was demonstrated. Treatment with allopurinol in BDL rats attenuated both ROS and BE, suggesting systemic oxidative stress is implicated in the pathogenesis of BE. Analysis of ROS and ammonia temporal resolution in the plasma of BDL rats suggests systemic oxidative stress might be an important "first hit", which, followed by increases in ammonia, leads to BE in chronic liver failure. In conclusion, chronic hyperammonemia and oxidative stress in combination lead to the onset of BE in rats with chronic liver failure.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is an autoimmune inflammatory disorder. Highly reactive oxygen free radicals are believed to be involved in the pathogenesis of the disease. In this study, RA patients were sub-grouped depending upon the presence or absence of rheumatoid factor, disease activity score and disease duration. RA Patients (120) and healthy controls (53) were evaluated for the oxidant—antioxidant status by monitoring ROS production, biomarkers of lipid peroxidation, protein oxidation and DNA damage. The level of various enzymatic and non-enzymatic antioxidants was also monitored. Correlation analysis was also performed for analysing the association between ROS and various other parameters.MethodsIntracellular ROS formation, lipid peroxidation (MDA level), protein oxidation (carbonyl level and thiol level) and DNA damage were detected in the blood of RA patients. Antioxidant status was evaluated by FRAP assay, DPPH reduction assay and enzymatic (SOD, catalase, GST, GR) and non-enzymatic (vitamin C and GSH) antioxidants.ResultsRA patients showed a higher ROS production, increased lipid peroxidation, protein oxidation and DNA damage. A significant decline in the ferric reducing ability, DPPH radical quenching ability and the levels of antioxidants has also been observed. Significant correlation has been found between ROS and various other parameters studied.ConclusionRA patients showed a marked increase in ROS formation, lipid peroxidation, protein oxidation, DNA damage and decrease in the activity of antioxidant defence system leading to oxidative stress which may contribute to tissue damage and hence to the chronicity of the disease.     

2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus)

2-Benzoxazolinone (BOA), a well-known allelochemical with strong phytotoxicity, is a potential herbicidal candidate. The aim of the present study was to determine whether phytotoxicity of BOA is due to induction of oxidative stress caused by generation of reactive oxygen species (ROS) and the changes in levels of antioxidant enzymes induced in response to BOA. Effect of BOA was studied on electrolyte leakage, lipid peroxidation (LP), hydrogen peroxide (H(2)O(2)) generation, proline (PRO) accumulation, and activities of antioxidant enzymes-superoxide dismutase (SOD, 1.15.1.1), ascorbate peroxidase (APX, 1.11.1.11), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6) and glutathione reductase (GR, 1.6.4.2) in Phaseolus aureus (mung bean). BOA significantly enhanced malondialdehyde (MDA) content, a product of LP, in both leaves and roots of mung bean. The amount of H(2)O(2), a product of oxidative stress, and endogenous PRO increased many-fold in response to BOA. Accumulation of PRO, MDA and H(2)O(2) indicates the cellular damage in the target tissue caused by ROS generated by BOA. In response to BOA, there was a significant increase in the activities of scavenging enzymes SOD, APX, GPX, CAT, and GR in root and leaf tissue of mung bean. At 5 mM BOA, GR activity in roots showed a nearly 22-fold increase over that in control. The present study concludes that BOA induces oxidative stress in mung bean through generation of ROS and upregulation of activities of various scavenging enzymes.     

Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells.

BACKGROUND: The oxidative status of cells has been shown to modulate various cell functions and be involved in physiological and pathological conditions, including hereditary chronic anemias, such as thalassemia. It is maintained by the balance between oxidants, such as reactive oxygen species (ROS), and antioxidants, such as reduced glutathione (GSH). METHODS: We studied peripheral RBC derived from normal and thalassemic donors. Flow cytometric methods were used to measure (1) generation of ROS; (2) the content of reduced GSH; and (3) peroxidation of membrane lipids as an indication of membrane damage. RESULTS: ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC, both at baseline as well as following oxidative stress, such as exposure to hydrogen peroxide. To simulate a state of iron overload, normal RBC were exposed to extracellular ferric ammonium citrate or hemin, or their Hb was denatured by phenylhydrazine. All these treatments increased ROS and lipid peroxidation and decreased GSH. These effects were reversed by N-acetyl cysteine, a known ROS scavenger. CONCLUSIONS: Flow cytometry can be useful for measuring oxidative stress and its effects on RBC in various diseases and for studying various chemical agents as antioxidants.     

Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage

Overexpression of bcl-2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl-2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl-2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron-enriched primary hippocampal cultures, with overexpression of bcl-2 driven by a herpes simplex virus amplicon: (i) Bcl-2 protected strongly against glutamate, whose toxicity is at least partially ROS-dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl-2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate-induced oxidative damage; (ii) Bcl-2 was also mildly protective against the pro-oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl-2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl-2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl-2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.     

Kidney cell death in inflammation: The role of oxidative stress and mitochondria

Pyelonephritis is an infectious disease, and common treatment strategy is based on antibiotic therapy directed at the elimination of a pathogen. However, urinary tract infections are accompanied by inflammation and oxidative stress, which are major damaging factors, and therefore can serve as a target for therapeutic intervention. The goal of this study was to clarify the role of the mitochondrial reactive oxygen species (ROS) in kidney cell damage under experimental pyelonephritis. We investigated the mechanisms of inflammation and the role of mitochondria and oxidative stress in inflammation in kidney tissue using in vivo and in vitro models of pyelonephritis. We observed the development of oxidative stress in renal tubular epithelium in vitro, and resulting apoptotic cell death. This oxidative damage was caused by the leukocytes producing ROS after interaction with bacterial antigens. The essential role of mitochondria-mediated oxidative stress was confirmed using an experimental model of pyelonephritis in vivo. We revealed increased levels of malonic dialdehyde in kidneys of rats with experimental pyelonephritis that pointed to lipid peroxidation. Besides, high ROS levels were observed in blood leukocytes from rats with pyelonephritis. The mitochondria-targeted antioxidant SkQ1 significantly reduced the signs of kidney inflammatory injury, in particular the infiltration of neutrophils. Summarizing the data obtained, we assume the importance of mitochondrial ROS in different phases of acute pyelonephritis onset. Protection of kidney cells from infection-mediated damage can be attained by the induction of tolerance mechanisms and by antioxidant treatment.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Modulation of experimental herpes encephalitis-associated neurotoxicity through sulforaphane treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection.     

Enhanced thioredoxin,glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H₂O₂‐induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade‐mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG‐mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.