Wojciech J. Piotrowski

Effect of Paraquat Intoxication and Ambroxol Treatment on Hydrogen Peroxide Production and Lipid Peroxidation in Selected Organs of Rat

Paraquat (Pq) is a herbicide which is very toxic to all animals and to man. It generates free radicals and leads to acute or chronic lung injury and usually to death. So far, the role of lipid peroxidation of cell membranes in the mechanism of its toxicity has not been proved satisfactorily and therefore in the present study we examined the concentration of hydrogen peroxide (H2O2) and various lipid peroxidation products (LPP)such as conjugated dienes (CD), lipid hydroperoxides (LH), malonyldialdehyde (MDA) and Schiff bases in selected organs of the rat given a single intraperitoneal dose 35 mg kg−1 Pq. We also evaluated the influence of a mucolytic and probably antioxidant drug, ambroxol, on Pq‐induced changes in the concentration of H2O2 and LPP. Paraquat increased the hepatic concentration of H2O2, CD, LH and MDA by approximately fourfold. Though the dose of Pq was nearly twice the LD50 dose, we did not notice any changes in the concentration of these substances in the critical organ, lung or heart and kidney. Ambroxol alleviated the increase of H2O2 in the liver but did not reduce the concentration of LPP. Moreover, the drug administered alone induced lipid peroxidation in the liver. Our results indicate that Pq does not induce H2O2 production and lipid peroxidation in the lung but it increases the concentration of H2O2 and LPP in the liver. Ambroxol inhibits the Pq‐induced increase in the concentration of H2O2 in the liver without protecting it against lipid peroxidation. Moreover, the drug alone may act as a pro‐oxidant.

Exhaled eicosanoids and biomarkers of oxidative stress in exacerbation of chronic obstructive pulmonary disease.

Introduction Eicosanoids and oxidants play an important role in inflammation, but their role in chronic obstructive pulmonary disease (COPD) is uncertain. In this study we hypothesized that levels of exhaled leukotrienes, prostaglandins and biomarkers of oxidative stress are increased in infectious exacerbations of COPD and that they decrease after antibiotic therapy. Material and methods Cysteinyl-leukotrienes (LTs), leukotriene B4 (LTB4), prostaglandin E4, hydrogen peroxide (H2O2) and 8-isoprostane were measured in exhaled breath condensate (EBC) in 16 COPD patients with infectious exacerbations (mean age 64 ±12 years, 13 male) on day 1, during antibiotic therapy (days 2-4), 2-4 days after therapy and at a follow-up visit when stable (21-28 days after therapy). Results There was a significant fall in concentration of cys-LTs, LTB4 and 8-isoprostane at visit 3 compared to day 1 (cys-LTs: 196.5 ±38.4 pg/ml vs. 50.1 ±8.2 pg/ml, p < 0.002; LTB4: 153.6 ±25.5 pg/ml vs. 71.9 ±11.3 pg/ml, p < 0.05; 8-isoprostane: 121.4 ±14.6 pg/ml vs. 56.1 ±5.2 pg/ml, p < 0.03, respectively). Exhaled H2O2 was higher on day 1 compared to that at visits 2 and 3 (0.74 ±0.046 µM vs. 0.52 ±0.028 µM and 0.35 ±0.029 µM, p < 0.01 and p < 0.01, respectively). Exhaled PGE2 levels did not change during exacerbations of COPD. Exhaled eicosanoids and H2O2 in EBC measured at the follow-up visit (stable COPD) were significantly higher compared to those from healthy subjects. Conclusions We conclude that eicosanoids and oxidants are increased in infectious exacerbations of COPD. They are also elevated in the airways of stable COPD patients compared to healthy subjects.

Correlation between eicosanoids in bronchoalveolar lavage fluid and in exhaled breath condensate

Exhaled breath condensate (EBC) has been increasingly used as a new and non-invasive method to study airway inflammation. In this study we have compared the concentrations of lipid mediators in EBCwith concentrations in bronchoalveolar lavage fluid (BALF). We included 37 patients undergoing bronchoscopy (12 sarcoidosis, 12 COPD, 6 lung cancer, 5 chronic cough, 1 Wegener’s granulomatosis, 1 sclerodermia). Patients were not allowed to have exacerbation or any change in concomitant medication for at least 4 weeks prior to the study. In all patients, EBC was collected immediately prior to the bronchoscopy. The levels of cys-LTs, LTB4, 8-isoprostane were significantly higher in BALF compared to EBC (p < 0.0001, p < 0.001, p < 0.0001 for cys-LTs, LTB4, 8-isoprostane respectively). Moreover, there was a strong positive correlation between both leukotriene B4 and 8-isoprostane in BALF and EBC (r = 0.53 and r = 0.79, p < 0.01, respectively) in patients with sarcoidosis and COPD but there was no correlation between eicosanoids BALF and EBC in patients with chronic cough and lung cancer. This is the first study to compare EBC and BALF in different lung diseases which demonstrated significant correlations between the levels of eicosanoids in BALF and EBCin patientswith COPD and sarcoidosis. EBC may be useful inmeasuring inflammation in several inflammatory lung diseases.

Rhinosinusitis in COPD: symptoms, mucosal changes, nasal lavage cells and eicosanoids

The coexistence of upper airways disease with chronic obstructive pulmonary disease (COPD) is not well documented. The aim of this research was to assess sino-nasal inflammation in COPD by various tools, and look for the impact on quality of life, relation to smoking, disease severity and systemic inflammation. Current and ex-smokers with COPD (n = 42) and healthy never-smokers (n = 21) were included in this study. COPD severity was assessed by GOLD criteria and BODE index. Markers of systemic inflammation were measured. Nasal symptoms and general quality of life were assessed using the questionnaires; sino-nasal questionnaire (SNAQ-11) and St. Georges Respiratory Questionnaire (SGRQ). Nasal endoscopy and saccharine test were performed. Nasal lavages were collected for cytological examination and eicosanoids (cysteinyl leukotrienes, leukotriene B4, 8-isoprostane). Symptoms and endoscopic scores were higher in COPD (P ≤ 0.0001). Only SGRQ symptoms subscore correlated with SNAQ-11 (r = 0.34, P = 0.035). Mucociliary clearance was impaired only in current smokers (9.91 ± 0.49 versus 13.12 ± 0.68 minutes, P ≤ 0.001). 8-isoprostane was higher in COPD smokers compared to the controls (0.17 ± 0.04 versus 0.34 ± 0.09 pg/g protein, P < 0.05). Endoscopic score and mucociliary of impairment patients who currently smoked cigarettes correlated with concentrations of 8-isoprostane. None of the parameters correlated with disease severity and markers of systemic inflammation. We provide evidence of upper airways disease in COPD, which appears to be related more to patients who currently smoke than to disease severity.

The selected genetic polymorphisms of metalloproteinases MMP2, 7, 9 and MMP inhibitor TIMP2 in sarcoidosis

Summary Background Increased activity of metalloproteinases may play a role in the initiation and propagation of inflammation in sarcoidosis, and may also be one of the factors responsible for the development of lung fibrosis. The aim of this study was to verify whether polymorphisms of MMP2 C-735T, MMP7 A-181G, MMP9 T-1702A and tissue inhibitor of metalloproteinase (TIMP)2 G-418C predispose to sarcoidosis. Material/Methods The study included 139 patients with sarcoidosis and 100 healthy subjects. MMPs and TIMP2 mRNA were measured in peripheral blood lysate using real-time RT-PCR. DNA for genetic polymorphism was extracted from peripheral blood by GTC method. Protein concentrations in peripheral blood lysates were measured by ELISA, and MMP2 and 9 activities in BAL fluid were estimated by gel zymography. Results TT genotype in MMP9 T-1702A was more frequent in sarcoidosis (p<0.0001, OR=13.71, 95% CI 7.02–26.80) and resulted in higher expression of MMP9 mRNA (p<0.0001). No differences were found between TT and AT/AA patients in terms of radiological stage, lung function test parameters, activity markers and the presence/absence of Löfgren syndrome. There were no differences in the distribution of MMP2, MMP7 and TIMP2 polymorphisms. Messenger RNAs, as well as protein concentrations of MMP2, 7, 9, and TIMP2 were elevated in patients with sarcoidosis (p<0.0001 for each). Conclusions The TT homozygotes of MMP9 T-1702A genotype may be predisposed to sarcoidosis. Elevated MMP2, 7, 9, and TIMP2 mRNAs suggest their inducibility.

Release of hydrogen peroxide by rat type II pneumocytes in the prolonged culture.

Type II pneumocytes (T II pneumocytes) produce hydrogen peroxide (H(2)O(2)), which may be potentially dangerous for the lung. These cells in culture differentiate to type I-like pneumocytes and it may reflect the differentiation which follows the injury of alveolar epithelium. This work was undertaken to estimate the H(2)O(2) release by T II pneumocytes, freshly isolated and cultured up to 8 days. The light and electron microscopy evaluation confirmed the differentiation of T II pneumocytes to type I-like cells. The release of H(2)O(2), estimated spectrofluorimetrically as homovanillic acid oxidation product obtained in the presence of horseradish peroxidase, was significantly higher at day 4 (0.63+/-0. 68nmol/mg protein/min, P</=0.02) and 6 (0.46+/-0.31, P</=0.001) compared to fresh cells (0.15+/-0.08). Phorbol esters increased H(2)O(2) release at day 2 (0.39+/-0.22 vs 0.16+/-0.08, P</=0.02) and the inhibition of protein kinase C resulted in the decrease at day 2 (0.14+/-0.06 vs 0.07+/-0.02, P</=0.025), day 6, (0.49+/-0.25 vs 0. 15+/-0.08, P</=0.005) and 8 (0.76+/-0.63 vs 0.23+/-0.29, P</=0.02). Inhibition of intracellular catalase resulted in a significant increase only at day 2 (0.23+/-0.1 vs 0.15+/-0.09, P</=0.05). Inhibition of mitochondrial respiratory chain decreased H(2)O(2) release at day 2 (0.13+/-0.11 vs 0.07+/-0.07, P</=0.002) and 4 (0. 75+/-0.88 vs 0.61+/-0.85, P</=0.002). These results indicate that alveolar epithelium may be a source of potentially dangerous ROS and that the cell differentiation is accompanied by the increase of H(2)O(2) production. Both mitochondrial respiratory chain and membrane-bound NADPH-oxidase may be responsible for the production of H(2)O(2) by T II pneumocytes.

Exhaled 8-isoprostane as a prognostic marker in sarcoidosis. A short term follow-up.

Background8-Isoprostane (8-IP) is a marker of lipid peroxidation. Elevated concentrations have been reported in BAL fluid and exhaled breath condensate (EBC) in sarcoidosis (S). To validate the prognostic value of this marker we tested whether: 1. high initial EBC 8-IP predispose to more severe disease; 2. low initial concentrations increase a chance of early remission; 3. remissions are connected with the decrease of EBC 8-IP.Methods40 patients (S) have been examined initially (V1) and after 8.5 ± 0.5 months (V2). EBC 8-IP concentrations were measured by ELISA. Chest X-ray, lung function test, serum ACE and Ca2+ concentrations, 24 hrs Ca2+loss, abdominal ultrasonography, symptoms evaluation were performed.ResultsWe confirmed higher concentrations of 8-IP in EBC of patients with sarcoidosis (p = 0.001). Relative risk (RR) of persistence of disease at V2 when initial 8-IP was above 20 pg/mL was 1.04, and the frequency distributions estimated by χ2 test were not significantly different. A chance (RR) of early complete remission when V1 8-IP was below DL, was 3.33 (p = 0.04 by χ2 test). A significant decrease of 8-IP at V2 was observed only in patients who received treatment (p = 0.03), but not in those with spontaneous remission.ConclusionsWe come to the conclusion, that low initial 8-IP may be a positive prognostic factor. A decrease of 8-IP in treated patients reflects a non-specific effect of treatment and is not related to mere regression of disease.

Exhaled breath 8-isoprostane as a marker of asthma severity.

Introduction Oxidative stress is a non-specific feature of airway inflammation in asthmatics. 8-Isoprostane (8-IP), a prostaglandin-F2α isomer, is a relatively new marker of oxidative stress and may be measured in exhaled breath condensate (EBC) of patients with asthma. This research study aimed to evaluate the usefulness of EBC 8-IP as a marker of severity and control of severe adult asthma. Material and methods Twenty-seven severe, never-smoking asthmatics were studied. According to positive or negative reversibility testing, this group was subdivided into reversible and irreversible asthma groups. All participants were observed for 8 weeks during which they completed daily diary observations including day and night symptoms, number of awakenings, peak expiratory flow (PEF) variability, daily rescue medication usage and oral steroids consumption. They attended the clinic 3 times and on these occasions spirometry assessments, EBC collection and asthma control tests (ACT) were done. Two control groups were included: 11 healthy never-smokers and 16 newly diagnosed and never-treated, non-smoking mild asthmatics. Results There were no statistically significant differences between severe asthma and healthy control or never-treated asthma groups in concentrations of EBC 8-IP (median and interquartile range: 4.67; 2.50-27.92 vs. 6.93; 2.5-12.98 vs. 3.80; 2.50-10.73, respectively). No correlations were found between EBC 8-IP and asthma control parameters, such as ACT results, night and day symptoms, consumption of rescue medication, percentage of days free of oral steroids, PEF diurnal variation, lung function test results, forced expiratory volume in the 1 s reversibility, and markers of systemic inflammation. Conclusions Our study results suggest that EBC 8-IP measurements are not useful for asthma monitoring.

The Role of Mitochondria and Oxidative/Antioxidative Imbalance in Pathobiology of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common preventable and treatable disease, characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. The major risk factor of COPD, which has been proven in many studies, is the exposure to cigarette smoke. However, it is 15–20% of all smokers who develop COPD. This is why we should recognize the pathobiology of COPD as involving a complex interaction between several factors, including genetic vulnerability. Oxidant-antioxidant imbalance is recognized as one of the significant factors in COPD pathogenesis. Numerous exogenous and endogenous sources of ROS are present in pathobiology of COPD. One of endogenous sources of ROS is mitochondria. Although leakage of electrons from electron transport chain and forming of ROS are the effect of physiological functioning of mitochondria, there are various intra- and extracellular factors which may increase this amount and significantly contribute to oxidative-antioxidative imbalance. With the coexistence with impaired antioxidant defence, all these issues lead to oxidative and carbonyl stress. Both of these states play a significant role in pathobiology of COPD and may account for development of major comorbidities of this disease.

TGF-β and SMADs mRNA Expression in Pulmonary Sarcoidosis

Lung fibrosis is a complication of sarcoidosis, in which TGF-β/Smad pathway may play an important role. We evaluated gene expression of TGF-β1, SMAD2, 3 and 7 in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94) to better understand the mechanisms of sarcoid inflammation. The relative gene expression was analyzed by qPCR method. Selected clinical/radiological features and biochemical markers were taken into account in the analysis. We found that TGF-β1 and SMAD3 expressions in PB lymphocytes were significantly higher in sarcoidosis patients. Up-regulation of SMAD7 (inhibitory Smad) and down-regulation of SMAD3 in BAL cells in all subgroups were found. The expression of TGF-β1 in PB lymphocytes was the highest in patients with lung parenchymal involvement and in the insidious onset phenotype. The expression of TGF-β1 in BAL cells was higher in patients with abnormal spirometry (p=0.012), and TGF-β1 and SMAD3 in patients with restrictive pattern (p=0.034 and 0.031, respectively). Several statistically significant negative correlations were found between the expression levels of SMAD2 and 3 in BAL cells and various LFT parameters. We conclude that TGF-β/Smad pathway is involved in the pathogenesis of pulmonary sarcoidosis. These biomarkers (especially TGF-β1, SMAD2 and 3) are of a negative prognostic value.