Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.     

Systemic oxidative and antioxidative status in Chinese patients with asthma

BACKGROUND: Patients with asthma generate an increased amount of reactive oxygen species from peripheral blood cells. Reactive oxygen species produce many of the pathophysiologic changes associated with asthma and may contribute to its pathogenesis. OBJECTIVE: We investigated changes in antioxidant enzyme activities and oxidized glutathione (glutathione disulfide; GSSG) levels in erythrocytes from a group of healthy control Chinese subjects (n=135) and patients with asthma (n=106). METHODS: Baseline pulmonary function was measured for all subjects. Antioxidant status was evaluated by measuring erythrocyte superoxide dismutase, catalase, and glutathione peroxidase activities. Oxidative stress was also measured in terms of GSSG in erythrocytes with a kinetic microassay. RESULTS: Patients with asthma had significantly increased erythrocyte superoxide dismutase and catalase activities compared with controls (61.10 +/- 1.30 U/g hemoglobin [Hb] vs 55.51 +/- 1.82 U/g Hb [P=.018] and 0.0637 +/- 0.0021 U/g Hb vs 0.0257 +/- 0.0120 U/g Hb [P <.001] for the asthma and control groups, respectively). Conversely, erythrocyte glutathione peroxidase activity decreased (44.21 +/- 1.33 mU/g Hb vs 50.07 +/- 1.39 mU/g Hb for the asthma and control groups, respectively; P=.003). Patients with asthma also had significantly higher GSSG levels in erythrocyte hemolysates compared with controls (167.40 +/- 2.93 micromol/L vs 44.98 +/- 0.44 micromol/L for the asthma and control groups, respectively; P <.001), indicating increased oxidative stress. CONCLUSIONS: Asthma is accompanied by an alteration in systemic antioxidant status due to possible oxidative stress in this disease.     

Effects of pioglitazone on hyperglycemia-induced alterations in antioxidative system in tissues of alloxan-treated diabetic animals.

Hyperglycemia not only generates reactive oxygen species but also attenuates antioxidant mechanisms creating a state of oxidative stress. Oxidative stress is thought to play a crucial role in pathogenesis of chronic diabetic complications. Pioglitazone is a new oral antidiabetic agent, a potent inhibitor of glycation and potent antioxidant. In the present study, normoglycemic and alloxan-induced diabetic rabbits were treated with pioglitazone (1 mg/kg daily) for 4 and 8 weeks. At the end, glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione (GSH) and protein carbonyl groups (PCG) were evaluated in homogenates of liver and kidney. Chronic hyperglycemia caused a marked increase in oxidative processes and some changes in activity of antioxidants. In liver, diabetic vs. control values (mean+/-S.E.M; P<0.05) for GSH-Px were 181.0+/-5.4 vs. 203.1+/-1.9 and 187.4+/-6.6 vs. 240.9+/-18.8mU/mg protein. Pioglitazone treatment for 8 weeks affected GSH-Px activity in diabetic liver (261.5+/-7.3 mU/mg protein). In diabetic kidney, GSSG-R activity (20.6+/-1.6 vs. 32.4+/-1.5 and 23.6+/-0.6 vs. 36.3+/-0.3 mU/mg protein) and GSH level (16.6+/-0.5 vs. 23.2+/-0.9 and 17.9+/-0.5 vs. 23.2+/-0.6 nmol/mg protein) were diminished, while PCG level (0.32+/-0.03 vs. 0.11+/-0.02 and 0.35+/-0.03 vs. 0.16+/-0.03 nmol/mg protein) was elevated. In diabetic kidney, pioglitazone restored to control values GSSG-R activity (34.4+/-1.4 and 30.6+/-0.1 mU/mg protein) as well as GSH (25.5+/-1.2 and 21.6+/-0.5 nmol/mg protein) and PCG (0.16+/-0.01 and 0.19+/-0.02 nmol/mg protein) levels. The present study showed that pioglitazone reduced to some extent the oxidative stress enhanced by chronic hyperglycemia.     

Clinical biochemical evaluation of central fatigue with 24-hour continuous exercise

OBJECTIVE: The present study was conducted to clarify the effects of ultra-marathon (ultra long-term aerobic exercise in which people run long distances) on the brain; examine the issue of central fatigue; verify the serotonin hypothesis of exercise-induced brain fatigue, and ascertain relationships between central fatigue and oxidative stress. METHODS: Subjects consisted of 15 individuals (12 men, 3 women) who ran continuously for 24 h. Mean age was 44 +/- 9 years (range, 31 approximately 64 years). Blood tests were conducted: (1) before starting to run (around 09:00); (2) 16h after starting (02:00 the next day); and (3) just after the finish (around 10:00 the next day) to measure the serum levels of serotonin, melatonin, free tryptophan (f-Tp) and free fatty acid. At the same time, urine samples were collected to measure levels of urinary biopyrrins (BPn). Subjective symptoms were investigated using the Japanese version of the Profile of Mood States (POMS) instrument. RESULTS: (1) Participants ran a mean (+/- SD) distance of 162.6 +/- 18.3 km. (2) There were not marked changes in serum serotonin levels. Serum melatonin levels at 3 time points were 3.4 +/- 0.6 pg/ml, 57.2 +/- 15.2pg/ml and 7.8 +/- 8.9pg/ml, respectively(p < 0.01 before start vs. 16h after start). Serum f-Trp levels at the 3 time points were 5.4 +/- 0.9 nmol/ml, 9.7 +/- 2.1 nmol/ml and 11.5 +/- 4.9 nmol/ml, respectively (p< 0.05 before start vs. just before finish). Free fatty acid levels were 0.42 +/- 0.10 nmol/ml, 1.26 +/- 0.11 nmol/ml and 1.39 +/- 0.23 nmol/ml, respectively (p < 0.01 before start vs. 16 hours after start) (p < 0.05 before start vs. just after finish). (3) Urinary BPn levels increased with time, from 1.2 +/- 0.7 nmol/ml to 2.6 +/- 1.0 nmol/ml to 4.0 +/- 1.5 nmol/ml, respectively (p < 0.01 before the start vs. 16 hours after the start). (4) In terms of POMS scores, fatigue score (Factor F) increased, but vitality score (Factor V) was high at all time points and did not demonstrate any marked changes. Scores for anger and hostility were low (Iceberg profile-type: convex type). Urinary BPn levels were correlated significantly with both serum f-Trp level and Factor F:(y = 8.41x + 2.5, r = 0.708, n = 42) and (y = 2.82x + 5.9, r = 0.568, n = 42), respectively. Urinary BPn thus reflected the degree of subjective fatigue with a high level of sensitivity. CONCLUSIONS: The present results suggest that running continuously for 24h induces brain fatigue and that oxidative stress may be involved.     

Oxidative stress and enzymatic antioxidant status in patients with nonalcoholic steatohepatitis

Oxidative stress is an important pathophysiological mechanism in nonalcoholic steatohepatitis (NASH). To assess whether there are relationships between oxidative stress and antioxidant enzymes in the development of NASH, we investigated oxidative stress by measuring serum malondialdehyde (MDA) and nitric oxide (NO) and antioxidant status by measuring serum glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and superoxide dismutase (SOD). The study included 18 patients (13 men, 5 women; mean age 42 yr) with biopsy proven NASH and 16 healthy volunteers (10 men, 6 women; mean age 38 yr). Serum levels of MDA, NO, GSH, GSH-Px, GR and SOD were determined by spectrophotometric methods. Serum levels (mean +/- SD) of MDA (6.7 +/- 1.6 vs 2.8 +/- 1.7 nmol/ml, p 0.0001), NO (135 +/- 28 vs 113 +/- 35 mmol/L, p 0.04), GSH (919 +/- 137 vs 770 +/- 128 mmol/L, p 0.003) were increased in patients with NASH vs controls. Serum levels of GSH-Px (1063 +/- 152 vs 1000 +/- 94 U/L) and GR (47 +/- 22 vs 40 +/- 21 U/L) were not singnificantly different in the patients vs controls. However, the serum level of SOD (1.24 +/- 0.32 vs 1.51 +/- 0.37 U/ml, p: 0.04) was significantly decreased. Impaired antioxidant defense mechanisms may be an important factor in the pathogenesis of NASH. Treatment approaches that affect the antioxidant enzymes may be beneficial in patients with NASH.     

Ageing alters aortic antioxidant enzyme activities in Fischer-344 rats.

Oxidative stress imposed by reactive oxygen species is now believed to contribute to hypertension, atherosclerosis and ageing of the vasculature all involving a loss of relaxation. The antioxidant enzymes glutathione peroxidase, superoxide dismutase and catalase play a crucial role in defending against the ravages of oxidative stress. Our purpose was to characterize age-related changes in glutathione peroxidase, superoxide dismutase and catalase in the rat aorta. Aortas were extracted from seven young (4 months), seven middle aged (18 months) and seven old (24 months) animals. Analysis of variance was used with Fisher-LSD post hoc to determine mean differences among glutathione peroxidase, superoxide dismutase and catalase. Aortic glutathione peroxidase activities rose steadily with age expressed in micromol mg protein-1 min-1 +/- SEM (young: 141 +/- 22; middle aged: 198 +/- 18; old: 229 +/- 26) reaching significance between young and old. Superoxide dismutase activities significantly decreased in middle aged when compared with young (young: 22 +/- 2 vs. middle aged: 15 +/- 2 U mg protein-1) before trending upward again in old age (19 +/- 2). Catalase activities dropped significantly between young and old when expressed in mU mg protein-1 (young: 230 +/- 30; middle aged: 173 +/- 18; old: 144 +/- 23). Ratios for the various enzymes indicate a shrinking contribution of catalase with ageing, with an enhanced role for glutathione peroxidase in the antioxidant defence. These data in aortas of ageing rats show a complex alteration of the antioxidant profile.     

Structural and functional transitions of the drug-metabolising systems under oxidative injury.

Ionizing radiation damage to cells is mainly due to the action of very reactive hydroxyl radicals, excited states and free radicals of macromolecules. It represents a class of chemical assaults dependent on the formation in high yields of highly reactive organic radicals. The aim of this work was to study the radiation-induced damage of rat liver microsomal membranes. Rats were singly irradiated with gamma-rays (1 Gy), sacrificed after 1 to 4 days and liver microsomal membranes were isolated. The level of lipid peroxidation products in the microsomal membranes increased (0.59+/-0.05 nmol TBARS/mg protein in comparison with 0.45+/-0.03 nmol/mg protein for the control), whereas the rates of NADPH-oxidation by liver microsomal membranes (1.26+/-0.13 nmol/min/mg protein in comparison with 1.99+/-0.21 nmol/min/mg protein for the control) and NADPH-ferricyanide reduction (168+/-11 nmol/min/mg protein in comparison with 269+/-15 nmol/min/mg protein for the control) decreased after 1 day following the whole body irradiation. At the same time we did not observe any significant changes in the level of microsomal membranous protein SH groups after the irradiation. The TBARS level and the rate of NADPH-oxidation but not the activity of ferricyanide NADPH-reductase were brought back to the control values 4 days after irradiation. The susceptibility of microsomal membranes to the chemically induced oxidative stress (by exogenous organic hydroperoxide (tBHP) treatment) before and after the whole body irradiation of rats was compared. The post-mortal liver microsomal membrane treatment by tBHP drastically changed the membrane structure and enzymatic activities. The TBARS level highly increased and the protein SH-group content decreased after chemically induced oxidative stress. The microsomal membrane rigidity increased after tBHP treatment up to 0.5 mM and slowly decreased at higher oxidant concentrations. These changes were more significant and occurred at lower oxidant concentrations in the microsomes of the irradiated animals after 1 day as compared to the control ones. The microsomal NADPH-oxidase and Fe(3+)-NADPH oxidoreductase activities decreased after the tBHP treatment of the microsomes of non-irradiated animals and either increased or remained unchanged for irradiated rats. Thus, low-dose rat irradiation as well as the microsomal membrane oxidative agent treatment significantly changed the membrane functional properties. The preliminary irradiation increased the membrane susceptibility to the chemically induced oxidative stress.     

Tyrosine phosphatase SHP-1 in oxidative stress and development of allergic airway inflammation

Oxidative stress has been implicated in allergic responses. SHP-1 is a target of oxidants and has been reported as a negative regulator in a mouse model of asthma. We investigated the effect of oxidative stress on the development of allergic airway inflammation in heterozygous viable motheaten (mev/+) mice deficient of SHP-1. Wild-type (WT) and mev/+ mice were compared in this study. Human alveolar epithelial cells (A549) transfected with mutant SHP-1 gene were used to evaluate the role of SHP-1 in lung epithelial cells. Hydrogen peroxide (H(2)O(2)) and Paraquat were used in vitro and in vivo, respectively. We also investigated whether mev/+ mice can break immune tolerance when exposed to aeroallergen intranasally. Compared with WT mice, bronchoalveolar lavage (BAL) cells and splenocytes from mev/+ mice showed a different response to oxidant stress. This includes a significant enhancement of intracellular reactive oxygen species and STAT6 phosphorylation in vitro and increased CCL20, decreased IL-10, and increased number of dendritic cells in BAL fluid in vivo. Mutant SHP-1-transfected epithelial cells secreted higher levels of CCL20 and RANTES after exposure to oxidative stress. Furthermore, break of immune tolerance, as development of allergic airway inflammation, was observed in mev/+ mice after allergen exposure, which was suppressed by antioxidant N-acetylcystein. These data suggest that SHP-1 plays an important role in regulating oxidative stress. Thus, increased intracellular oxidative stress and lack of SHP-1 in the presence of T helper cell type 2-prone cellular activation may lead to the development of allergic airway inflammation.     

Effects of oxygen metabolites on rat alveolar type II cell viability and surfactant metabolism

Neutrophil-derived reactive oxygen metabolites have been implicated as one mechanism for the cellular injury in the adult respiratory distress syndrome. Previous studies have demonstrated that alveolar lung fluid of patients with adult respiratory distress syndrome has abnormal composition and surface active properties. To examine the effects of oxygen metabolites on the viability and metabolism of type II alveolar pneumocytes, the cellular source of surfactant, isolated rat type II pneumocytes were exposed to reactive oxygen metabolites generated by the enzymatic action of xanthine oxidase upon hypoxanthine. Utilizing a 51Cr release assay to detect cellular death, we found that oxygen metabolites were lethal to type II cells in a dose-dependent manner. To demonstrate that oxygen metabolites were responsible for the toxicity, we assessed the protective effects of catalase and superoxide dismutase, scavengers of hydrogen peroxide and the superoxide anion, respectively. At a xanthine oxidase concentration of 50 mU/ml, catalase reduced the percentage of 51Cr release from 58.9 +/- 3.1% (SEM) to 7.2 +/- 2.3% (p less than 0.0001), whereas superoxide dismutase was without protection (58.9 +/- 3.1% versus 54.2 +/- 1.8% (p greater than 0.05). To determine whether oxygen metabolites also impair surfactant metabolism, we measured the incorporation of [3H]palmitate into the surfactant component disaturated phosphatidylcholine by type II pneumocytes. We found that sublethal amounts of generated oxygen metabolites caused a progressive decrease in the amount of [3H]palmitate incorporated into disaturated phosphatidylcholine. For example, using a xanthine oxidase concentration of 5 mU/ml (which causes no increased 51Cr release), we found that [3H]palmitate incorporation into disaturated phosphatidylcholine fell from a control level of 3.53 +/- 0.22 X 10(5) to 0.66 +/- 0.10 X 10(5) dpm/10(6) cells/4 hours (p less than 0.0001). Both catalase and superoxide dismutase protected the [3H]palmitate incorporation of oxygen metabolite-exposed type II cells. We conclude that reactive oxygen metabolites are injurious to type II pneumocytes and may result in impaired surfactant synthesis even at sublethal doses. Thus, oxygen metabolites generated by stimulated phagocytic cells may be responsible in part for the decreased surfactant that has been observed in adult respiratory distress syndrome.     

Glucose‐6‐phosphate dehydrogenase inhibition attenuates acute lung injury through reduction in NADPH oxidase‐derived reactive oxygen species

Acute lung injury (ALI) is a heterogeneous disease with the hallmarks of alveolar capillary membrane injury, increased pulmonary oedema and pulmonary inflammation. The most common direct aetiological factor for ALI is usually parenchymal lung infection or haemorrhage. Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) are thought to play an important role in the pathophysiology of ALI. Glucose‐6‐phosphate dehydrogenase (G6PD) plays an important role both in production of ROS as well as their removal through the supply of NADPH. However, how G6PD modulation affects NOX2‐mediated ROS in the airway epithelial cells (AECs) during acute lung injury has not been explored previously. Therefore, we investigated the effect of G6PD inhibitor, 6‐aminonicotinamide on G6PD activity, NOX2 expression, ROS production and enzymatic anti‐oxidants in AECs in a mouse model of ALI induced by lipopolysaccharide (LPS). ALI led to increased G6PD activity in the AECs with concomitant elevation of NOX2, ROS, SOD1 and nitrotyrosine. G6PD inhibitor led to reduction of LPS‐induced airway inflammation, bronchoalveolar lavage fluid protein concentration as well as NOX2‐derived ROS and subsequent oxidative stress. Conversely, ALI led to decreased glutathione reductase activity in AECs, which was normalized by G6PD inhibitor. These data show that activation of G6PD is associated with enhancement of oxidative inflammation in during ALI. Therefore, inhibition of G6PD might be a beneficial strategy during ALI to limit oxidative damage and ameliorate airway inflammation.     

Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.     

Heparin coating of the extracorporeal circuit combined with leukocyte filtration reduces coagulation activity, blood loss and blood product substitution

BACKGROUND: Cardiopulmonary bypass is associated with activation of the coagulation cascade. Occasionally, this results in postoperative hemorrhage and consequently the need for blood products associated with increasing costs and risk of infection. Contact activation of the intrinsic coagulation pathway, and damage to cellular blood components with the release of proteolytic substances from neutrophil granulocytes have been linked to these coagulation disorders. METHODS: Eighteen routine CABG-patients were randomly assigned to totally heparin coated circuits (Bioline coating) combined with leukocyte filtration in a double blind protocol (group I), 34 patients served as controls (group II). Leukocyte filters were activated before release of the aortic crossclamp. Coagulation activity, postoperative blood loss, and substitution with blood products were assessed. RESULTS: Blood loss in the first 24h after surgery was significantly lower with combined application of heparin coating and leukocyte filters (group I) vs. controls (group II) (526+/-78 ml vs. 786+/-88 ml; p<0.05). Thrombin formation represented by prothrombin fragments 1+2 was significantly lower in group I vs. group II after declamping of the aorta (2.1+/-0. 3 nmol/L vs. 4.0+/-0.3 nmol/L; p<0. 05). Group I showed higher AT II plasma than group II (48.8+/-3.2% vs. 41.5+/-1.77%; p<0.05). CONCLUSIONS: Leukocyte filtration during reperfusion in heparin coated cardiopulmonary bypass circuits is associated with lower coagulation activation, decreased blood loss and reduced transfusion of packed red cells in elective CABG patients.     

Reduction of macrophage activation after antioxidant enzymes gene transfer to rat insulinoma INS-1 cells

BACKGROUND: After transplantation, islet damage occurs through oxidative stress and host immune rejection mediated in part by macrophage activation. We investigated the influence of the overexpression of catalase (CAT) and Cu/Zn superoxide dismutase (Cu/Zn SOD) by rat insulinoma INS-1 beta cells exposed to oxidative stress on their viability and murine macrophage activation. METHODS: INS-1 cells were infected with adenoviral vectors containing CAT (AdCAT) or Cu/Zn SOD (AdSOD) genes. After 72 hours, noninfected and infected INS-1 cells were exposed to oxidative stress and their viability was assessed using a colorimetric assay. Murine peritoneal exudate macrophages (mPEM) incubated with the supernatant of infected and stressed INS-1 cells were tested for chemotaxis and cytokine release (TNF-alpha, IL-alpha and IFN-gamma). RESULTS: After infection, AdCAT and AdSOD gene transfer protected INS-1 cells from the toxicity of different oxidative reagents. The exposure of non-infected INS-1 cells to oxidative stress stimulated mPEM chemotaxis. INS-1 cells infection with AdCAT or AdSOD reduced significantly mPEM chemotaxis from 2.41 +/- 0.31 to 1.61 +/- 0.17 and from 2.53 +/- 0.24 to 1.27 +/- 0.14 respectively (n = 5; p < 0.05). Cytokine release by mPEM was stimulated after exposure to stressed noninfected INS-1 cell supernatant. CAT and Cu/Zn SOD overexpression by infected INS-1 cells decreased significantly the release of TNF-alpha from 268.18 +/- 30.18 to 81.40 +/- 23.58 pg/ml and from 446.96 +/- 75.47 to 20.37 +/- 2.38 pg/ml respectively (n = 6; p < 0.001). The overexpression of these enzymes also reduced significantly the release of IL-1beta and IFN-gamma. CONCLUSIONS: CAT or Cu/Zn SOD gene transfer to INS-1 cells preserved them from oxidative damage and reduced the macrophage activation induced by these pancreatic cells. Therefore, protection of pancreatic beta cells against oxidative injury by antioxidant enzymes gene transfer is an effective approach to overcome the deleterious actions of macrophages in pancreatic islet transplantation.     

Carbonic anhydrase II and alveolar fluid reabsorption during hypercapnia

Carbonic anhydrase II (CAII) plays an important role in carbon dioxide metabolism and intracellular pH regulation. In this study, we provide evidence that CAII is expressed in both type I (AECI) and type II (AECII) alveolar epithelial cells by RT-PCR and Western blotting in freshly isolated rat cells. These results were further confirmed by double immunostaining with CAII antibodies and AECI- or AECII-specific markers in freshly isolated alveolar epithelial cells and rat lung tissues. Inhibition of CAII by acetazolamide or methazolamide delayed the decrease in the intracellular pH observed during hypercapnia in cultured AECI, AECII, and AECI-like cells. In an isolated-perfused rat lung model, alveolar fluid reabsorption significantly decreased during high CO(2) exposure, which was not prevented by carbonic anhydrase inhibition. Thus, we provide evidence that CAII is expressed in rat alveolar epithelial cells and does not regulate lung alveolar fluid reabsorption during hypercapnia.     

Hypertrophic alveolar type II cells from silica-treated rats are committed to DNA synthesis in vitro

Alveolar type II cell hyperplasia and hypertrophy are common reparative responses of the alveolar epithelium after silica-induced lung injury. We studied in vitro DNA synthesis in type II cells isolated after silica instillation in the rat to determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis. To determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation, we also examined the mitogenic effect of bronchoalveolar lavage fluid (BALF) from silica-treated rats on type II cells in primary culture. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal silica instillation, cultured in DME supplemented with 10% fetal bovine serum, and labeled with [3H]thymidine from day 1 to day 3 in culture. DNA synthesis was determined by [3H]thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 +/- 5.4 x 10(3) dpm/well 7 d after silica instillation to 34.4 +/- 5.0 x 10(3) dpm/well at 28 d. Type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (greater than 88%) were the same in all groups of elutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 +/- 2.8 x 10(3) dpm/well) than the normotrophic type II cells (11.1 +/- 0.7 x 10(3) dpm/well) [P less than 0.01]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of GSTM1 and GSTT1 polymorphism with lipid peroxidation in benign prostate hyperplasia and prostate cancer: a pilot study

Association of glutathione S-transferase (GST) M1 and T1 deletions with benign prostate hyperplasia (BPH) and prostate cancer is well reported. These enzymes metabolize numerous toxins thus protecting from oxidative injury. Oxidative stress has been associated with development of BPH and prostate cancer. The present study was designed to analyze role of GST deletions in development of oxidative stress in these subjects. GSTs are responsible for metabolism of toxins present in tobacco therefore effect of tobacco usage in study groups was also studied. Three groups of subjects: BPH (57 patients), prostate cancer (53 patients) and controls (46 subjects) were recruited. Genotyping was done using a multiplex polymerase chain reaction (PCR) method. Malondialdehyde (MDA) levels as marker of oxidative stress were estimated by measuring thiobarbituric acid reactive substance (TBARS) in plasma. Based on genotyping, subjects were categorized into: GSTM1+/GSTT1+, GSTM1-/GSTT1+, GSTM1+/GSTT1- and GSTM1-/GSTT1-. Significantly higher plasma MDA levels were noticed in GSTM1-/GSTT1- as compared to GSTM1+/GSTT1+ in all study groups. Double deletion (GSTM1-/GSTT1-) is associated with higher oxidative stress which might play a role in the pathogenesis of BPH and prostate cancer. However, other markers of oxidative stress should be analyzed before any firm conclusion.     

氧化应激状态的评价

机体代谢过程中活性氧不断地通过非酶促反应和酶促反应产生,每日约有1％-3％的摄入氧转变为超氧阴离子(superoxideanion,O2-)及其活性衍生物,但在抗氧化酶以及外源性和内源性抗氧化剂的协同作用下被不断清除,在生理情况下,ROS的生成     

Increased manganese superoxide dismutase protein in type II epithelial cells of rat lungs after inhalation of crocidolite asbestos or cristobalite silica.

Manganese-containing superoxide dismutase (Mn-SOD) is a mitochondrial enzyme implicated in cellular defense from oxidative damage. We investigated the immunocytochemical distribution and protein concentration of Mn-SOD in rat lungs in response to aerosolized crocidolite asbestos or cristobalite silica, fibrogenic minerals eliciting generation of oxidants by cellular and acellular pathways. Rats were exposed to 7-10 mg/m3 dust for 6 hours a day for 10 days. Experimental and sham control rats were euthanized 10 days after cessation of exposure, and lungs prepared for immunocytochemistry and determination of amounts of Mn-SOD protein. Quantitation of Western blots showed that the amount of immunodetectable Mn-SOD increased in lungs exposed to asbestos or silica by approximately 1.3- and 2.4-fold, respectively, when compared with sham controls. Immunoelectron microscopy using the protein A-gold technique showed that Mn-SOD was located predominantly in mitochondria of type II epithelial cells. Fibroblasts contained little immunodetectable Mn-SOD, whereas type I epithelial cells, polymorphonuclear leukocytes (PMNs), and endothelial cells contained no detectable protein. Some alveolar macrophages (AMs) were found with labeled mitochondria, whereas most interstitial macrophages had no detectable protein. Quantitative analysis of type II cells showed that the number of immunogold particles per unit of mitochondrial area increased in the terminal airways of lungs exposed to asbestos or silica by 2.2-fold and 3.6-fold, respectively, over controls. Morphometric analyses indicated that the size of type II cells, as well as the number of interstitial macrophages and PMNs, increased in the terminal respiratory tissue of silica-exposed lungs. Less pronounced histopathologic changes were evident in asbestos-exposed lungs. These results indicate that the relative concentration of Mn-SOD increases preferentially in type II epithelial cells subsequent to inhalation of silica or asbestos. The magnitude of induction of Mn-SOD protein in these cells and whole lung correlated with the inflammatory potential of these minerals.