Tadeusz Pietras

Increased content of hydrogen peroxide in the expired breath of cigarette smokers

Cigarette smoking causes an influx of mononuclear phagocytes and polymorphonuclear leucocytes into the lower airways. These cells have altered oxygen metabolism and release more H2O2 than phagocytes from nonsmokers. In this study, we intended to determine whether asymptomatic cigarette smokers exhale more H2O2 than healthy nonsmokers. The content of H2O2 in the expired condensate of 27 nonsmokers and 33 cigarette smokers was measured spectrofluorimetrically (homovanillic acid method). The mean H2O2 level in the expired breath condensate of all cigarette smokers was about fivefold higher than that found in the whole nonsmoker group (0.24 +/- 0.32 versus 0.05 +/- 0.11 nM). However, only 16 smokers (49%) and 6 nonsmokers (22%) had detectable levels of H2O2 in expired breath that reached values 0.49 +/- 0.28 and 0.23 +/- 0.10 nM, respectively. Although the cigarette smoking status was similar for both male and female smokers, females expired 2.5 fold less H2O2 than males (0.15 +/- 0.24 (n = 21) versus 0.38 +/- 0.39 (n = 12) nM. No correlation was found between expired H2O2 levels and cigarette smoking status expressed as the daily cigarette consumption, cumulative cigarette consumption and urinary cotinine concentration. It is suggested that in some smokers, expressed H2O2 can be a noninvasive marker of oxidant overload in the lower airways related to cigarette smoking.

Antioxidant properties of ambroxol

We tested whether Ambroxol, a drug which stimulates the release of surfactant by pneumocytes type II, may also possess antioxidant properties. To assess the reactivity of Ambroxol with reactive oxygen species, we analysed its ability to decompose hydrogen peroxide (H2O2) and to inhibit the superoxide (O2.-)-dependent autooxidation of pyrogallol, hydroxyl radical (.OH)-mediated deoxyribose oxidation, and hypochlorous acid (HClO-induced chlorination of monochlorodimedon. Ambroxol was found to be a sufficient scavenger of HClO and .OH and also revealed the capacity to decompose H2O2. At concentrations of 25 and 70 microM, it inhibited HClO-induced chlorination of monochlorodimedon by 22 +/- 13 and 59 +/- 14%, respectively. Similarly, at concentrations of 1, 2, and 10 mM, Ambroxol decreased .OH-mediated deoxyribose oxidation by 47 +/- 11, 75 +/- 9, and 89 +/- 4%. In addition, at concentrations of 1 to 5 mM, it completely protected linoleic acid from .OH-induced peroxidative damage. Ambroxol had a weak effect on O2.(-)-dependent autooxidation of pyrogallol. Our results indicate that Ambroxol has antioxidant activity, which may have clinical significance in protecting lung tissue from oxidant-induced injury.

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.

Protective effect of ambroxol against heat- and hydrogen peroxide-induced damage to lung lipids in mice

We wanted to determine whether ambroxol, a drug which stimulates the release of surfactant by type II pneumocytes, can protect lung lipids from peroxidative damage in mice. Animals were injected intraperitoneally with ambroxol, 0.169 mmol.kg-1, or 1 ml buffer once a day for three consecutive days. Lipid peroxidation was then induced in lung homogenates either by means of heat, 50 degrees C, or H2O2, 10 mmol.l-1. The lung homogenates from ambroxol-treated animals revealed decreased lipid peroxidation in response to both stimuli. The heat- and H2O2-induced generation of conjugated dienes (a first lipid peroxidation product) in ambroxol-treated lung homogenates was 3.7 and 3.1 fold lower than in the lungs from buffer-injected mice. Ambroxol, as an inhibitor of heat- and H2O2-induced lipid peroxidation, was equipotent to and stronger than the two antioxidants, N-acetylcysteine and methionine, respectively. Ambroxol was not able to protect heart and liver lipids. These results suggest that ambroxol can sufficiently enhance the antioxidant defence in lung tissue and can act as a lung lipid antioxidant.

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.

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice

The influence of lipopolysaccharide fromEscherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2°C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0,9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.

Analysis of two polymorphisms of the manganese superoxide dismutase gene (Ile-58Thr and Ala-9Val) in patients with recurrent depressive disorder

Reactive oxygen species (ROS) may contribute to the pathogenesis of depressive disorder (DD). Functional genetic polymorphisms of manganese superoxide dismutase (MnSOD) are candidates for DD susceptibility. The study examined the relationship between MnSOD gene polymorphisms (Ala-9Val, Ile-58Thr) and DD in the Polish population. The association study was conducted in a case-control design in DD patients (n=149) and healthy controls (CG; n=149) by genotyping. Assessment of Ala-9Val genotype distribution and disease odds ratio demonstrated a statistically significant difference between the compared groups only in the female subgroup. The obtained results suggest a role of the MnSOD polymorphism in the development and course of depression.

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.

The N363S and I559N single nucleotide polymorphisms of the h-GR/NR3C1 gene in patients with bronchial asthma

Bronchial asthma is a disease of multifactorial etiology. The natural variability of the DNA sequence within the h-GR/NR3C1 gene affects both the conformation and the activity of glucocorticoid receptors. There are 2 major types of resistance to glucocorticoids (GCS)-resistant asthma failing to respond to treatment with high doses of inhaled and oral glucocorticoids. Type I GCS-resistant asthma is cytokine-induced or acquired. Type II GCS resistance involves generalized primary cortisol resistance, which affects all tissues and is likely to be associated with a mutation in the glucocorticoid receptor (GCR) gene or in genes that modulate GCR function. There are clear examples of glucocorticoid gene h-GR/NR3C1 polymorphisms that can influence responses and sensitivity to glucocorticosteroids. Among the numerous polymorphisms observed within this gene, N363S and I559N single nucleotide polymorphisms (SNPs) may play an important role in the development of bronchial asthma and in the alteration of sensitivity to GCS in severe bronchial asthma. The aim of this research project was to study the correlation between the N363S and I559N polymorphisms of the h-GR/NR3C1 gene and the occurrence of asthma in a population of Polish asthmatics. Peripheral blood was obtained from 210 healthy volunteers and 234 asthma patients. Structuralized anamnesis, spirometry and allergy skin prick tests were performed in all participants. Genotyping was carried out using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and PCR-HRM methods. In the healthy, non-atopic population, the GG variant of the N363S polymorphism was found with a 5.7% frequency. In asthma patients, GG SNP of N363S occurred with the frequency of 6.4%. In the groups of patients with uncontrolled moderate asthma and uncontrolled severe disease, the genotype distribution for the investigated polymorphisms were as follows: N363S, AA, AG, GG occurring with 0.8750/0.0834/0.0416 frequency and I559N, TT, TA, AA occurring with 1.000/0.000/0.000 frequency. The analysis demonstrated a significantly higher frequency of the A and G variants of the N363S polymorphisms in uncontrolled moderate asthma and uncontrolled severe disease than in the healthy population. No variant-related differences in the frequency of the studied I559N polymorphism were demonstrated in healthy controls and asthma patients. In conclusion, the N363S polymorphism of the h-GR/NR3C1 gene is significantly associated with an increased sensitivity to glucococorticoids in vivo and susceptibility to the development of a moderate to severe form of uncontrolled bronchial asthma in the Polish population. This observation needs to be confirmed in a larger group of subjects.

Anxiety, depression and methods of stress coping in patients with nicotine dependence syndrome

Summary Background Nicotinism is the most common addiction in Poland. Nicotine dependence is the cause of numerous behavioral diseases, including ischemic heart disease, neoplasms and chronic obstructive pulmonary disease. A question arises whether a tendency to anxiety and depressive reactions, as well as the strategies of coping with stressful situations, is involved in the clinical presentation of this addiction. Material/Methods The study was conducted in a group of 88 nicotine addicts without serious systemic comorbidities and in 84 healthy subjects. All the participants were assessed with Beck Depression Inventory (BDI), Spielberger State-Trait Anxiety Inventory (STAI) and the Coping Inventory for Stressful Situations (CISS). Results The mean intensity of anxiety as a trait and anxiety as a state, as well as its level, were found to differ between the groups (Sten 6.28±1.52 and 4.86±1.05, p=0,0000 for the trait, and 6.09±1.25 and 4.92±1.29, p=0.0000, for the state, respectively). Similarly, depression was demonstrated to be more intensive in nicotine addicts than in healthy subjects (12.76 points ±4.77 vs. 10.76±4.83, p=0.007). Among the 5 scales assessed by CISS, smokers demonstrated higher prevalence of emotion-oriented coping than controls (standard 9 6.27±1.70 in smokers vs. 5.67±1.57, p=0.019) and involvement in distracting activities (5.84±1.48 vs. 5.28±1.46, p=0.014). Conclusions The obtained results indicate that anxiety and depression, as well as differences in coping with stress situations, distinguish nicotine addicts from non-smokers.