ROCK activation in lung of idiopathic pulmonary fibrosis with oxidative stress.

The Rho-associated coiled-coil containing protein kinase, (Rho-kinase or ROCK) undergoes activation by oxidative stress. ROCK-II, which is an isoform of ROCK, is activated in a murine model of lung fibrosis. The present study evaluated the level of oxidative stress and activation of ROCK-II in patients with idiopathic pulmonary fibrosis (IPF). The ROCK-II level and the phosphorylation of myosin phosphatase subunit-1 (p-MYPT-1), a hallmark of ROCK activation, were examined by immunohistochemistry of lung tissue sections. The 8-iso prostaglandin-F2alpha (8-isoPGF2alpha) level, as a marker of oxidative stress, of exhaled breath condensate was significantly higher in IPF patients than in control patients. In IPF lungs, ROCK-II was predominantly expressed by bronchial epithelial cells, as well as at a lower level by airway smooth muscle cells, vascular smooth muscle cells, and the fibroblasts of fibroblastic foci (FF). In addition, there was moderate p-MYPT-1 expression in these cells of IPF lungs. In control lungs, ROCK-II was expressed by these cells. p-MYPT-1 was weakly expressed by the bronchial epithelial cells. In conclusion, ROCK-II was activated in various lung cells of IPF patients along with oxidative stress detected by 8-isoPGF2alpha elevation. The ROCK pathway may play a role in the development of IPF oxidative stress.

Gastrointestinal symptoms in idiopathic pulmonary fibrosis patients treated with pirfenidone and herbal medicine.

Pirfenidone is an antifibrotic agent for patients with pulmonary fibrosis, but this drug has adverse gastrointestinal (GI) effects. The first aim of this study was to assess GI symptoms due to pirfenidone by using a new questionnaire for reflux symptoms and dismotility symptoms. Whether adding herbal medicine of rikkunshi-to improved GI symptoms due to pirfenidone therapy was also investigated. This was a randomized controlled trial performed on 17 IPF patients. The patients were assigned to two groups, and the study period was 8 weeks. The pirfenidone group received pirfenidone therapy for 8 weeks with add-on rikkunshi-to from 4 weeks, while the control group did not receive either of these agents. To assess the effects of RK, plasma levels of acyl-ghrelin and des-acyl-ghrelin, serum KL-6 and surfactant protein-D, and pulmonary function tests were monitored. GI symptoms were most severe during the initial 2 weeks of pirfenidone therapy at a dose of 600 mg/day. Both reflux symptoms and dismotility symptoms deteriorated. Rikkunshi-to improved GI symptoms to the level prior to pirfenidone therapy. Plasma levels of des-acyl-ghrelin and acyl-/des-acyl-ghrelin ratio changed significantly at 8 weeks compared to 2 weeks. GI adverse events due to PFD were most severe in the first 2 weeks of treatment at a dose of 600 mg/day, and both reflux and dismotility symptoms deteriorated, but the drug was well tolerated at 1200 mg/day. Rikkunshi-to contributed to improvement of GI symptoms, but plasma ghrelin levels did not reflect the improvement of GI symptoms.

Chronic exposure to particles caused bronchioloalveolar carcinoma in a patient with cryptogenic organizing pneumonia evaluated by elemental analysis.

An 81-year-old Japanese man had organizing pneumonia (OP), and he had worked as a painter and had a history of exposure of various paints over 20 years. The major features on computed tomography (CT) in patients were cryptogenic organizing pneumonia (COP) showing airspace consolidation, and air bronchograms were consistent finding in consolidation in right lung of S¹°. Such parenchymal abnormalities were clinically and pathologically diagnosed COP and the lesion was improved by corticosteroid therapy. About 1.5 years later, similar shadows emerged in new locations of right S4 and left S8, and these were bronchioloalveolar carcinoma (BAC) classified as adenocarcinoma. BAC causes similar X-ray changes to COP and inflammation accompanying BAC can also respond to corticosteroids, which may lead to delay in the diagnosis of BAC associated with COP. These radiological features lead to difficulty in making a diagnosis of new parenchymal diseases. The present patient had been painter, and metals of carcinogens were proven in both tissue of COP and BAC. Here, we reported a painter with COP and new-onset BAC who had been exposed to particles proven by elemental analysis. The combination of COP with BAC is considered uncommon, but the risk of BAC may increase when there is a history of particle inhalation.

Intracellular glutathione redox status in human dendritic cells regulates IL-27 production and T-cell polarization.

BACKGROUND: Glutathione redox status, changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione, plays a significant role in various aspects of cellular function. In this study, we examined whether intracellular glutathione redox status in human dendritic cells (DCs) regulates the polarization of Th1/Th2 balance. METHODS: Human monocyte-derived DCs (MD-DCs) treated with glutathione reduced form ethyl ester (GSH-OEt) or L-buthionine-(S,R)-sulfoximine (BSO) were stimulated by lipopolysaccharide (LPS), and the levels of polarization cytokines were measured. Next, DCs matured by LPS or thymic stromal lymphopoietin (TSLP) were cocultured with allogeneic CD4(+) naive T cells and Th1/Th2 balance was evaluated by cytokine production from the primed T cells. RESULTS: Monocyte-derived DCs exposed to GSH-OEt and BSO had increased and decreased intracellular GSH contents, respectively. Lipopolysaccharide-induced interleukin (IL)-27 production was enhanced by GSH-OEt and suppressed by BSO, but neither GSH-OEt nor BSO affected the expression of HLA-DR, CD80, CD83, or CD86. Mature GSH-OEt-treated MD-DCs enhanced interferon (IFN)-γ production from CD4(+) T cells compared with nontreated MD-DCs, and small interfering RNA (siRNA) against IL-27 suppressed the effect of GSH-OEt on IFN-γ production. Additionally, although human myeloid DCs activated by TSLP (TSLP-DCs) prime naïve CD4(+) T cells to differentiate into Th2 cells, treatment of TSLP-DCs with GSH-OEt reduced IL-13 production and enhanced IFN-γ production by CD4(+) T cells. Interleukin-27 siRNA attenuated the inhibitory effect of GSH-OEt on Th2 polarization. CONCLUSION: Our results reveal that Th1 and Th2 responses are controlled by intracellular glutathione redox status in DCs through IL-27 production.

Changes of immunomodulatory cytokines associated with omalizumab therapy for severe persistent asthma.

Omalizumab is an anti-IgE monoclonal antibody that was proven effective for the treatment of severe asthma. IgE plays a central role in allergic asthma, and an anti-allergic effect of omalizumab has been confirmed in terms of its impact on Th2 cytokines. The objective of the present study is to determine the influence of omalizumab on clinical parameters and circulating immuoregulatory cytokines. Patients with severe allergic asthma were enrolled and given four months of omalizumab therapy. Changes of symptoms and other parameters were assessed, including the asthma control test (ACT) score, morning peak expiratory flow (PEF), peripheral eosinophil count, total serum IgE, and pulmonary function tests. The use of corticosteroids and short-acting bronchodilators, as well as the number of unscheduled hospital visits, were monitored. Circulating levels of cytokines were analyzed with a multiplex cytokine immunoassay in patients with or without omalizumab therapy. Asthma symptoms (evaluated by the ACT score and morning PEF) improved with omalizumab treatment, while total IgE was elevated. Use of corticosteroids and short-acting bronchodilators and the number of unscheduled hospital visits for exacerbation of asthma were all reduced by omalizumab treatment. The level of macrophage inflammatory protein 1-δ (MIP1-δ) was significantly reduced after omalizumab therapy and was high in patients without omalizumab. IL-16 also tended to decrease with omalizumab therapy. Both MIP1-δ and IL-16 decreased as asthma improved over the 4-month period of omalizumab therapy. These findings suggest that omalizumab may act via IgE-mediated immunoregulation of MIP1-δ and IL-16.

Decreased interstitial FOXP3(+) lymphocytes in usual interstitial pneumonia with discrepancy of CXCL12/CXCR4 axis.

Regulatory T cells (Treg) play a critical role in immune homeostasis and expansion of Treg is controlled by chemokine receptors. The chemokine CXCL12 and its G-protein-coupled receptor (CXCR4) are involved in the development of idiopathic pulmonary fibrosis (IPF), but the association of Treg with the CXCL12/CXCR4 axis has not been documented. The aim of this study is to determine the distribution and extent of CXCL12/CXCR4 expression in idiopathic type of pulmonary fibrosis, and the relation of Treg expansion in the interstitium of pulmonary fibrosis patients to CXCL12/CXCR4 expression. CXCL12 expression was examined by immunostaining and ELISA in tissue specimens from patients with usual interstitial pneumonia (UIP, n=15), patients with fibrotic non-specific interstitial pneumonia (f-NSIP, n=4), and controls (n=6). CXCR4 expression was examined by in situ hybridization and immunoblotting. Expression of CD45, CD3, CD20, transcription factor forkhead box P3 (FOXP3), and CD25 was assessed by immunostaining. Fibrosis was evaluated by determining the established fibrosis (EF) score. The CXCL12/CXCR4 axis was upregulated in UIP and f-NSIP, and CXCL12 derived from lung tissue attracted CXCR4(+) cells. CXCR4(+) cells showed a CD3(+) cell distribution pattern. The interstitial FOXP3(+)/CD3(+) and CD25(+)/CD3(+) cell ratios were lower in UIP than f-NSIP, but the CXCR4(+)/CD3(+) cell ratio was not different. The FOXP3(+)/CD3(+) cell ratio and EF score were inversely correlated. These findings suggest that the CXCL12/CXCR4 axis contributes to inflammation in UIP and f-NSIP by promoting the accumulation CXCR4(+) lymphocytes, and a decrease of Treg is correlated with the severity of fibrosis in UIP.

Analysis on the co-localization of asbestos bodies and Fas or CD163 expression in asbestos lung tissue by in-air micro-pixe.

To prevent and control disease caused by exposure to various agents, it is necessary to determine the harmful level of intervention and to establish a method for measuring that level. In-air microparticle-induced X-ray emission (in-air micro-PIXE) analysis is based on irradiation of specimens with a proton ion microbeam, and has been modified for biological application. Two-dimensional analysis and quantitative analysis using the system confirmed that asbestos induced apoptosis by upregulating Fas expression and also revealed the accumulation of CD163-expressing macrophages in the lungs of patients with asbestosis. By quantitative comparison of the area of Fas or CD163 expression and the Fas- or CD163-negative area in asbestos lung tissue, the harmful levels which caused the expression of Fas or CD163 could be estimated on Silica, Ferrous iron, and Magnesium (the components of asbestos) deposition. These results indicate that the system could be useful for investigating the pathogenesis of inhaled particle-induced immune reactions and for determining harmful levels of exogenous agents.

Eosinophilic bronchiolitis indicating eosinophilic airway disease with overexpression of carcinoembryonic antigen in sinus and bronchiole: case report.

The present case showed eosinophilic bronchiolitis and sinusitis with an overexpression of carcinoembryonic antigen (CEA) in lung and sinus and an elevation of serum CEA level, both of which were improved by oral steroid therapy. A 54-year-old asthmatic woman had developed a shortness of breath on exertion, and the chest X-ray revealed diffuse centrilobular shadows. Her serum CEA level had increased gradually. Eosinophil infiltration and overexpression of CEA were demonstrated in both the lung and sinus by immunohistochemistry. Both the lung and sinus lesions, and the serum CEA level were improved by oral steroid therapy. No evidence of tumor was found by extensive examination. From this case, eosinophilic bronchiolitis was considered to be an airway disease like "eosinophilic sinobronchiolitis" through the common pathophysiology of CEA, and serum CEA level was a good marker of disease condition.

CXCR4+FOXP3+CD25+ lymphocytes accumulate in CXCL12-expressing malignant pleural mesothelioma.

CXCL12 is a chemokine that binds to a G-protein-coupled receptor (CXCR4). CXCL12 is expressed in various tumors and is considered as playing an important role in tumor growth and invasion. The aim of this study is to investigate the expression of CXCL12 in human malignant mesothelioma (MM), the chemotactic effect of CXCL12 derived from MM, and the expression of CXCR4 in MM tissues in relation to regulatory T cells. CXCL12 expression was examined by immunostaining of tissue specimens from malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPEM). The MM group comprised 6 patients (4 men/2 women, MPM=4, MPEM=2, aged 56.0 +/- 12.4 years) and the control (non-mesothelioma) group also had 6 patients (4 men/2 women aged 65.0 +/- 6.7 years). CXCL12 mRNA expression was also examined by RT-PCR in MPM cell lines (H28, H2052, and H2058), while CXCR4 mRNA expression was examined by in situ hybridization in MPM tissue. CXCL12 was expressed in the cytoplasm of MM cells from all patients, but was not expressed in the control group. H2052 and H2058 cells expressed CXCL12 mRNA, but H28 cells did not. CXCL12 in MM tissue homogenate supernatant had a chemotactic effect on CXCR4-expressing THP-1 cells. CXCR4 mRNA was expressed by a part of LCA+CD3+ Foxp3+CD25+ T cells that were located adjacent to the border of CXCL12-expressing epithelioid MPM. These findings suggest that CXCL12 contributed to tumor-related inflammation by inducing the accumulation of CXCR4-expressing cells with regulatory T cell markers around MM.

Hypoxemia with high alveolar-arterial oxygen gradient but no lung involvement in a patient with Churg-Strauss syndrome: case report.

Churg-Strauss syndrome (CSS) is characterized by asthma and/or a history of allergy, eosinophilia and an often life-threatening systemic necrotizing vasculitis. We describe a patient with CSS and hypoxemia with a high alveolar-arterial oxygen gradient (AaDO2), but no pulmonary parenchymal involvement. The patient also had a low diffusion capacity with normal lung volume and a high level of serum thrombomodulin, a marker of endothelial cell injury. Treatment for CSS, such as corticosteroid, improved both hypoxemia and AaDO2 consistent with amelioration of diffusion capacity and serum thrombomodulin level, suggesting that this pathosis involves microangiopathy with endothelial cell damage induced by vasculitis in pulmonary blood vessels.

In-air micro-particle induced X-ray emission analysis of asbestos and metals in lung tissue.

Inhalation of asbestos increases the risk of lung cancer and pulmonary fibrosis. It is difficult to directly assess the distribution and content of inhaled particles in lung tissue sections. The purpose of this study is to employ an in-air micro particle induced X-ray emission (in-air micro-PIXE) system for assessment of the spatial distribution and content of asbestos and other metals in lung tissue. A proton ion-microbeam from this system was applied to irradiate lung tissue of patients with or without asbestosis, tumor tissue from both groups, and asbestos fibers (in vitro). The content of each element composing asbestos and those of other metals were calculated and their distribution was assessed from the characteristic X-ray pattern for each element obtained after irradiation. This in-air micro-PIXE system could identify the location of asbestos bodies composed of Si, Mg, and Fe in lung tissue sections. Macrophage and lymphocytes accumulated in that area. This new system also revealed deposits of titanium, nickel, and cobalt in the lung tissues, in addition to asbestos bodies. The Si and Fe content were higher in lungs with asbestosis than in lungs without asbestosis or in tumor tissue. Analysis of asbestos fibers composed of chrysotile, crocidolite, and amosite showed that the ratios of Si, Fe, and Mg corresponded with those for the chemical structures. In-air micro-PIXE analysis is useful for assessing the distribution and quantities of asbestos bodies and also other metals in lung tissue comparing to immune-related cell localizations, and is also useful for analysis of standard asbestos fibers.

The relationship between oxidative stress and acid stress in adult patients with mild asthma.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of asthma. Interestingly, a low airway pH and a high concentration of 8-isoprostane, a marker of oxidative stress, has been reported to cause inflammatory airway diseases. However, the relationship between these 2 markers and pulmonary function has not been determined in mild asthma patients. METHODS: pH and 8-isoprostane concentration were measured in exhaled breath condensate (EBC) from patients with mild asthma (n = 44) and healthy subjects (n = 20). The relationship between acid stress (pH) and oxidative stress (8-isoprostane) was then analyzed, along with the relationships between these 2 markers and lung function. RESULTS: The median (interquartile range [IQR]) pH of EBC was significantly lower in asthma patients than in control subjects (7.53 [7.41-7.68] vs 7.70 [7.62-7.74], P < .05), while the median (IQR) 8-isoprostane concentration of EBC was significantly higher in asthma patients than control subjects (16.2 [11.7-19.1] vs 3.5 [2.6-7.9] pg/mL, P < .05). There was no correlation between pH and 8-isoprostane concentration. Furthermore, lung function was not correlated with either pH or 8-isoprostane concentrations in EBC. CONCLUSIONS: Acid stress and oxidative stress assessed by pH and 8-isoprostane concentration, respectively, in EBC did not show parallel changes associated with asthma and were not correlated with lung function in asthma patients. These 2 stress factors may have different roles in the pathogenesis of asthma.

Acute impact of volcanic ash on asthma symptoms and treatment.

Information about the impacts of disasters on health is useful for establishing hazard prediction maps and action plans of disaster management. This study aims at learning effective asthma management from the volcano disaster of Mount Asama eruption in Japan on September 1, 2004. We conducted a cross-sectional study to assess the acute impact of volcanic ash on asthma symptoms and their treatment changes by using a questionnaire completed by 236 adult asthmatic patients and their physicians. In the ashfall over 100g/m2 area, 42.9 percent of asthma patients suffered exacerbations, PEF decreased, asthma treatments increased, and inhalation of beta2 stimulants was used most for exacerbated asthma. Compared to severe asthma patients, mild and moderate asthma patients were most at risk. Severe asthma patients were not affected since most of them knew their asthma status was severe, and did not go outside and kept windows closed. Deteriorated asthma symptoms of wheezing, chest tightness and cough appeared in the ashfall over 100g/m2 area. Ash contained inhalable 10microm diameter particles, and included high concentrations of airway toxic substrates of silica. These data suggest that ashfall over 100 g/m2 is harmful, access to these areas by asthma patients needs to be restricted, and these areas need to improve asthma treatment. In addition, the increase in the proportion of asthma patients with wheeze and cough are diagnostic clues for ash-induced asthma in affected areas, and can be used by doctors to tell whether patients are receiving sufficient asthma treatment.

Manifesto on small airway involvement and management in asthma and chronic obstructive pulmonary disease: an Interasma (Global Asthma Association - GAA) and World Allergy Organization (WAO) document endorsed by Allergic Rhinitis and its Impact on Asthma (ARIA) and Global Allergy and Asthma European Network (GA2LEN).

Evidence that enables us to identify, assess, and access the small airways in asthma and chronic obstructive pulmonary disease (COPD) has led INTERASMA (Global Asthma Association) and WAO to take a position on the role of the small airways in these diseases. Starting from an extensive literature review, both organizations developed, discussed, and approved the manifesto, which was subsequently approved and endorsed by the chairs of ARIA and GA2LEN. The manifesto describes the evidence gathered to date and defines and proposes issues on small airway involvement and management in asthma and COPD with the aim of challenging assumptions, fostering commitment, and bringing about change. The small airways (defined as those with an internal diameter <2 mm) are involved in the pathogenesis of asthma and COPD and are the major determinant of airflow obstruction in these diseases. Various tests are available for the assessment of the small airways, and their results must be integrated to confirm a diagnosis of small airway dysfunction. In asthma and COPD, the small airways play a key role in attempts to achieve disease control and better outcomes. Small-particle inhaled formulations (defined as those that, owing to their size [usually <2 µm], ensure more extensive deposition in the lung periphery than large molecules) have proved beneficial in patients with asthma and COPD, especially those in whom small airway involvement is predominant. Functional and biological tools capable of accurately assessing the lung periphery and more intensive use of currently available tools are necessary. In patients with suspected COPD or asthma, small airway involvement must be assessed using currently available tools. In patients with subotpimal disease control and/or functional or biological signs of disease activity, the role of small airway involvement should be assessed and treatment tailored. Therefore, the choice between large- and small-particle inhaled formulations must reflect the physician's considerations of disease features, phenotype, and response to previous therapy. This article is being co-published in Asthma Research and Practice and the World Allergy Organization Journal.

Identification of novel mutations in the dihydropyrimidine dehydrogenase gene in a Japanese patient with 5-fluorouracil toxicity.

5-Fluorouracil (5-FU) is used widely in the treatment of several common neoplasms. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU. Several recent studies have described a pharmacogenetic disorder in which cancer patients with decreased DPD activity develop life-threatening toxicity following exposure to 5-FU. We reported recently the first Japanese case of decreased DPD activity accompanied by severe 5-FU toxicity. The present study describes the results of molecular analysis of this patient and her family, in which three novel mutations (Arg21Gln, Val335Leu, and Glu386Ter) of the gene coding for DPD were identified. We also revealed that Arg21Gln and Glu386Ter are on the same allele and that Val335Leu is on the other allele, on the basis of analysis of the family genome. Expression analysis in Escherichia coli showed that Val335Leu and Glu386Ter led to mutant DPD protein with significant loss of enzymatic activity and no activity, respectively. The Arg21Gln mutation, however, resulted in no decrease in enzymatic activity compared with the wild type. The present data represent the first molecular genetic analysis of DPD deficiency accompanied by severe 5-FU toxicity in a Japanese patient.

Interferon-gamma rescues TNF-alpha-induced apoptosis mediated by up-regulation of TNFR2 on EoL-1 cells.

Recent studies show that apoptosis is important for the resolution of chronic inflammation. Using a human myeloblastic leukemia cell line, EoL-1, we investigated the effect of interferon-gamma (IFN-gamma), which differentiates EoL-1 into monocyte/macrophage-like cells on Fas antigen (Fas)- and tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. Both TNF and anti-Fas monoclonal antibody (CH-11) induced apoptosis of EoL-1 cells. Pretreatment with IFN-gamma for 72 hours enhanced the CH-11-induced apoptosis with up-regulation of Fas. However, the treatment markedly inhibited the TNF-induced apoptosis. In flow cytometric analysis, EoL-1 expressed two types of tumor necrosis factor receptors (TNFR1 and TNFR2), and the expression of TNFR2 but not of TNFR1 was up-regulated significantly after the IFN-gamma treatment. The TNF-induced apoptosis was mimicked by a TNFR1 stimulating antibody (htr-9), and was reversed by a TNFR1 blocking antibody (H398). Although the TNFR1-mediated cytotoxic signal was not affected by IFN-gamma pretreatment, blocking TNFR2 by a specific antagonistic antibody (utr-1) canceled the inhibitory effect of IFN-gamma. In conclusion, TNF-induced apoptosis was mediated preferentially by TNFR1, and the anti-apoptotic effect of IFN-gamma was result from up-regulated TNFR2 in EoL-1 cell line. This cell line is a useful model to provide new insights into crosstalk among Fas/FasL-, TNF-, and IFN-gamma-mediated signaling.

Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle.

To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.

Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: a possible mechanism of injury to heart and skeletal muscle in hyperthyroidism.

This study was designed to determine if peroxidation of biomembrane lipid and the protective system can be modified by the change in oxidative metabolism induced by thyroid dysfunction. The free radical scavengers (i.e. cuprozinc cytosolic and mangano mitochondrial superoxide dismutases, glutathione peroxidase, and catalase), mitochondrial oxidative marker enzymes (cytochrome c oxidase and fumarase), and lipid peroxide were measured in liver, heart, soleus (slow oxidative), and extensor digitorum longus (fast glycolytic) muscles. Rats were rendered hyper- or hypothyroid for 4 weeks and then killed. Superoxide dismutases were detected by specific RIAs: catalase by polarography, and lipid peroxide by fluorimetry. Hypothyroid rats failed to grow, while hyperthyroid rats had hypertrophied hearts but no growth failure. An increase in lipid peroxide was observed in the soleus and heart muscles of hyperthyroid rats. This was accompanied by an increase in mitochondrial superoxide dismutase and oxidative markers. No such change was observed in either fast glycolytic muscle or liver. Glutathione peroxidase decreased in all tissues of hyperthyroid rats, and there was a parallel decrease in catalase in most tissues. On the other hand, hypothyroidism induced a reduction in oxidative markers and mitochondrial superoxide dismutase in heart and skeletal muscles, but only a marginal change in lipid peroxidation. The cytosolic superoxide dismutase did not change in relation to either oxidative metabolism or lipid peroxidation. These results suggest that the enhanced oxidative metabolism and decreased glutathione peroxidase in hyperthyroidism result in an increase in lipid peroxidation and, in slow oxidative and heart muscle, possible organ damage. No adverse reaction mediated by active oxygen species was found in hypothyroid rat tissues.

Antioxidants in the serum of children with insulin-dependent diabetes mellitus.

To determine whether alteration in serum antioxidant status is related to the increased oxidative stress as a cause of diabetic angiopathy, we measured both the antioxidant activity (AOA) and total peroxyl radical-trapping antioxidant parameter (TRAP), and their component individual antioxidants in serum of children with insulin-dependent diabetes mellitus (IDDM). The AOA was measured as the ability to inhibit lipid autoxidation in brain homogenates. TRAP was assayed as the ability to delay lipid peroxidation induced by an azo initiator. Antioxidants measured were ceruloplasmin, transferrin, and albumin as components of AOA; and ascorbic acid, uric acid, protein sulfhydryl, and alpha-tocopherol as components of TRAP. Serum AOA appeared to be decreased in the diabetics in relation to poor glycemic control, corresponding to the decrease in transferrin and albumin. Serum haptoglobin level was also decreased in the diabetics. Similarly, the directly measured TRAP value was decreased in the diabetic serum mainly due to the decreased contribution of unidentified chain-breaking antioxidants, despite the increase in ascorbic acid and alpha-tocopherol. The decrease in both types of antioxidant activity in the diabetic serum, as new findings, suggests that a defective serum antioxidant status contributes to the increased oxidative stress in IDDM.

Tissue differences in antioxidant enzyme gene expression in response to endotoxin.

The effect of endotoxin on antioxidant gene expression and antioxidant enzyme activity in homogenates of the heart, liver, and kidney from Sprague-Dawley rats was compared by quantitation of m-RNA and enzyme activities. Alterations in the message level for Cu-Zn superoxide dismutase (SOD), Mn SOD, and catalase varied with the tissue type, length of exposure to endotoxin, and dose of endotoxin. In general, endotoxin treatment reduced Cu-Zn SOD expression in the heart and liver, but had no noticeable effect in the kidney. Mn SOD message levels were increased in the heart and kidney but decreased in the liver. Catalase expression was reduced in the kidney and increased marginally in the heart and liver. With regard to enzyme activity, endotoxin treatment reduced Cu-Zn SOD activity in the heart, liver, and kidney. Mn SOD activity showed little change in the heart, but increased in the liver and, to a lesser extent, in the kidney. Catalase activity showed little change in the heart and kidney but was decreased at 12 h in the liver. The differing responses of tissues to the oxidant stress of endotoxin exposure should be considered when evaluating the effect of endotoxin on antioxidant enzymes.