INTRODUCTION:

Cigarette smoke, a complex mixture of more than 4000 identified carcinogenic constituents that including both free radicals in high concentration and chemical compounds, which rapidly convert to other reactive substances ¹. Mainstream and side stream gas-phase cigarette smoke contain about the 1×10¹⁶free radicals per cigarette (5 ×10¹⁴ free radicals per puff). These radicals are remarkably stable in gas phase, lasting for more than 5 minutes. Smoking causes inflammation regardless of the presence or absence of lung disease. In smokers with chronic obstructive pulmonary disease (COPD), the inflammatory response is enhanced. Intuitively, most would expect that inhaled smoke, by means of some combination of its more than 4000 particulate and volatile components, induces local effects and airway inflammation. Indeed, most studies have focused on analysis of sputum, bronchoalveolar lavage fluid, and lung tissue from patients who smoke to assess these effects where they occur ².

Cigarette smoke contains nicotine and nicotine like alkaloids, PAHs, Aldehyde compounds, Aromatic amines, Heterocyclic amines, Aza-arenes, N-Nitrosamines and Miscellaneous organic/ inorganic compounds, and these all makes cigarette smoke a complete carcinogenic and cytotoxic mixture³. Cigarette smoke contains >4000 compounds and about 60 of them are animal carcinogenic and carcinogenic to human, also.

Benzo[a]pyrene (PAH), Nitroso-alkaloids, Carbonyl compounds etc. are potent prototype carcinogens, present in cigarette smoke at high concentration⁴. Some of the carcinogen from the CS like PAH’s and nicotine like alkaloids undergo metabolic activation and form adducts with cellular components (DNA, RNA and Cellular enzymes) or cause alkylation/ nitrosation of cellular components³. The mitochondrial respiratory chain is oneof the most important sites of ROS production under physiologicalconditions.Mitochondria-derivedROS are vital not only because mitochondrial respiratory chaincomponents are present in almost all eukaryotic cells but alsobecause the ROS produced in mitochondria can readily influencemitochondrial function without having to cope with long diffusiontimes from the cytosol⁵. The role of mitochondrial anti-oxidant enzymes in smoking was not studied so far. In this paper we, investigated whether and how the mitochondrial antioxidant enzymes changes in smoking injury. In smoking injury, we found the occurrence of alveolar vacuolization with matrix swelling. Further, decrease oxidative enzymes and increase of LPO level in smoking group.

Materials and Methods:

Animals and Experimental model

Mature Wistar rats weighing 150-250 g were procured from Laboratory Animal Resource, Division of Animal Genetics, IVRI, Izatnagar and acclimatized to laboratory condition at Animal House IFTM, at Moradabad at room temperature 24±2°C with a 12h/12h/light/dark cycle and 70% RH Twelve Wistar rats were divided into sham-operated control group (I) (n = 6) were kept in fresh air environment. Smoking group (II) (n= 6) was exposed for 60 minutes every day in an inhalation chamber (10 liters) for the two week (14 days). All rats were treated in accordance with the guideline for the Care and Use of Laboratory Animals (NIH Publication No.86-23, revised 1985) with the permission of institute ethical committee.

Fig.1. Shows mitochondrial antioxidant enzymes in sham operated control group and smoking groups.

Results are expressed as mean ± SD (n=6).

Tissue procurement:

Blood samples were obtained from the right ventricle via a left anterior thoracotomy at the time of sacrifice. A portion of the lung was fixed in buffered 10% formalin, embedded in paraffin, and stained using hematoxylin and eosin (H and E). Another portion of lung tissue was kept in normal saline under deep frozen condition for determination of mitochondrial enzymes.

Estimation of Lipid Peroxidation (LPO):

Lipid peroxide measured as described by Jordan and Schenkman et al.⁶, 1982. Beriefly, to 100 µl separated epithelial cells in 0.1(M) phosphate buffer saline, 1ml of 28% trichloroacetic acid was added and centrifuge. Nine hundred µl of 1% thiobarbituric acid was added to 1 ml of supernatant and volume was adjusted to 3 ml by of using phosphate buffer (pH 7.0), heated on boiling water for 60 min and cooled immediately. The absorbence was measured spectrophotometrically at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction coefficient of malondialdehyde (MDA) (1.56×10⁵) and expressed in terms of nmoles MDA/mg of protein.

Estimation of Mitochondrial Anti-oxidant Marker Enzymes:

Isolation of Lung Mitochondria:

Mitochondria were isolated as described by Starkov and Fiskum⁷, 2003. Beriefly,100 mg lung tissue was excised with ice-cold isolation buffercontaining 225 mM mannitol, 75 mM sucrose, 5 mM HEPES-KOH, pH7.4, 1 mM EGTA, and 1 gm L ^-1bovine serum albumin ina 15 ml Dounce homogenizer and homogenized manually with eightstrokes of pestle A followed by eight strokes of pestle B.The homogenate was diluted with 15 ml of isolation buffer, distributedinto four centrifuge tubes, and centrifuged at 3000 r.p.m. for4 min. The supernatant was separated and centrifuged again at14,000 r.p.m. for 10 min. The pellet was resuspended in 15 ml ofthe ice-cold isolation buffer without BSA and kept on ice, and30 µl of digitonin (10% stock solution in DMSO) was added.After 4 min incubation with occasional stirring by slow inversionof tubes, the suspension was diluted with 15 ml of ice-coldisolation buffer containing BSA and centrifuged at 14,000 xg for 10 min. The pellet was resuspended in 8 ml of ice-coldisolation buffer containing neither BSA nor EGTA and centrifugedagain at 14,000 r.p.m for 10 min. The final pellet containingmitochondria was resuspended in isolation buffer without EGTA or BSA toa concentration of 25-30 mg of protein ml^-1, stored in ice, and wasused within 5 h.

Fig .2(a-b). Shows histopathological Changes in lungs (x400). (a). Sham operated control rats. (b). Smoking groups

(a)

(b)

Estimination Reduced Glutathione (GSH):

Mitochondria GSH was estimated by as described by Habig et al⁸., 1974. Briefly, adding 0.2 ml mitochondrial enzyme to 2 ml distilled water followed by 3.0 ml of precipating mixture (1.67g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl and distilled water was added to make up to volume 100ml). Solution was centrifuged at 5000 x g for 5 min of supernatant 1 ml was added to 1.5 ml of phosphate buffer pH 7.4, followed by 0.5 ml DNTB reagent .The optical activity was measured at 412 nm using spectrophotometer. Absorbance of standard reduced GSH was noted and calculation was done.

Estimation Superoxide Dismutase (SOD):

Superoxide dismutase was determined as described by Hodgson and Fridovich et al.⁹, 1975. 50 ml of the mitochondrial enzyme was added to 75mM Tris-HCl buffer (pH 8.2), 30mM EDTA and 2mM of pyrogallol. Absorbance was recorded at 420nm at interval for 3 min by spectrophotometer. One unit enzyme activity is 50% inhibition of the rate of auto -oxidation of pyrogallol. The activity of SOD was expressed as units per mg of protein.

Fig. 3. Histopathological Changes after exposure Cigarette smoke

Estimination Catalase (CAT):

Catalase activity was determined in mitochondrial lysates using Aebi’s method with some modifications ¹⁰. 20 µl of the lysates was added to a cuvette containing 2 ml of phosphate buffer (pH7.0) and 1 ml of 30 mM hydrogen peroxide. Catalase activity was measured at 240 nm for 1 min using spectrophotometer .The molar extinction coefficient of H₂O_2, 43.6 M c.m^-1 was used to determine the catalase activity. One unit of activity is equal equivalent to one mM of H₂O₂degraded per minute and is expressed as units per mg of protein.

Histopathological evaluation by light microscope assay:

Serial of slice of the lung tissues were prepared from rat I each group and stained with hematoxylin-eosin (H and E) ad then observed the histological changes under light microscope at 100x and 400x magnification.

Statistical Analysis:

All values were expressed as mean± S.D. differences in mean values were compared using Mann-Whitney Test and Kruskal-Wallis Test (Nonparametric ANOVA).

RESULTS:

Activities of anti-oxidant enzymes viz. SOD, GSH and CAT were 1.28±0.027, 11.96±0.28 and 3.04±0.087 respectively in sham operated control rats which decreased to 0.87 ± 0.04, 8.31 ± 0.28 and 1.71±0.11 respectively in smoking group. LPO enzymes in sham operated control rats was 1.75±0.065 where as smoking group treated rats were increased to 3.23 ± 0.07 (Fig.1).

Expressed as nML^-1MDA mg^-1 of protein, Units per mg per 100 mg protein for SOD;mM L^-1 of 1-chloro 2,4-dinitrobenzene (CDNB) conjugated min^-1 100 mg^-1 protein for GST; mM L^-1 of H₂O₂ decomposed per min per mg protein for CAT.

Morphological Evaluation Light Microscope Assay:

Lung histopathology was evaluated based on sinusoidal congestion, cytoplasmic vacuolization, alveolar necrosis, and neutrophil infiltration. Smoking alveolar caused necrosis and sinusoidal congestion in Group II. There was mostly sparing of the periportal areas with progressive injury in the mid zonal and pericentral areas. Further hypoxemic cell death, which, and hypoxic vasoconstriction characterized by idiopathic pulmonary fibrosis (IPF) (UIP pattern), and pulmonary Langerhan's cell histiocytosis (eosinophilic granuloma of the lung) also observed (Fig. 2 and 3).

Discussion:

The mitochondrial respiratory chain is an important source of non-enzymatic generation of reactive species. During oxidative phosphorylation process, electrons are transferred by electron carriers NADH and FADH₂, through four complexes in the inner mitochondrial membrane to oxygen during generation of ATP¹¹.Under normal conditions, ^•O₂^-is immediately eliminated by natural defense mechanisms. The liver injury is mediated by free radicals or by the depletion of endogenous pool of antioxidants from mitochondria. Reactive species can be eliminated by a number of enzymatic and non- enzymatic antioxidant mechanisms. Generations of reactive species of I/R injury are NOS, NADPH oxidase and xanthine oxidase¹². Oxidant stress-inducedcell damage associated with oxidation of pyridine nucleotides in nucleus, accumulationof calcium in mitochondria as well as super oxide formation in mitochondrialeads to formation of membrane permeability transitionpores and breakdown of the mitochondrial membrane potential ¹³.

Earlier study showed that two sources are caused increased oxidative stress in lung tissue which is oxidants present in inhaled cigarette smoke, and the reactive oxygen species produced by the excessive number of inflammatory cells in the lung. Using lipid peroxidation as a marker, investigators have reported increased levels of isoprostane 14 in the exhaled breath and of 4-OH-nonenal 15 in the lungs of chronic smoker. Cigarette smoke also decreases histone deacetylas 16 which under normal conditions down regulates transcription of inflammatory genes. Conforming with earlier reported work, the present work showed that the enhanced production of nitric oxide through administration of L-arginine reduces oxygen free radicals, increases the mitochondrial antioxidants, increases respiratory marker mitochondrial enzymes and subsequently decreases the amount of lipid peroxidation by removing substrate for free radical generation by physiological mechanisms. Oxidant stress induced cell killing involves oxidation of pyridine nucleotides, accumulation of calcium ions in mitochondria, and superoxide formation by mitochondria, ultimately leading to formation of membrane permeability transition pores and breakdown of the mitochondrial membrane potential 17.

In present studies, the ultra structural changes demonstrated microscopically in smoking injured rats which showed severe degeneration of alveolar architecture. Further smoking group showed lung showed swollen cells with marked vacuolization.

Conclusively, the present findings suggest that smoking causes alveolar structure with severe degeneration of lung epithelial tissue. The pathophysiological and biochemical finding confirms that depletion of antioxidant enzymes during smoking which causes formation supra-oxide radicals formation and leads to injury.

Acknowledgement:

The authors are thankful to Prof. A.K Wahi, Dean, College of Pharmacy, IFTM for continuous support and encouragement. The authors are also thankful to Management, IFTM for roviding Post Graduate Institutional Research Grant.

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