Activated charcoal fi lter prevents emphysema 217

Activated charcoal fi lter effectively reduces p-benzosemiquinone from the mainstream and prevents emphysema

NEEKKAN DEY, ARCHITA DAS, ARUNAVA GHOSH and INDU B CHATTERJEE* Department of Biotechnology and Dr B C Guha Centre for Genetic Engineering and Biotechnology, University College of Science, Kolkata 700019, *Corresponding author (Fax, 91-033-24614849; Email, [email protected]) In this paper, we have made a comparative evaluation of the cytotoxicity and pathophysiological effects of mainstream smoke from cellulose acetate (CA)-fi ltered with that of charcoal-fi ltered cigarettes developed in our laboratory. Previously, we had demonstrated that the mainstream smoke from an Indian CA-fi ltered commercial cigarette contains p-benzosemiquinone (p-BSQ), a major, highly toxic, long-lived water-soluble radical. Here, we have examined 16 brands of different CA-fi ltered cigarettes including Kentucky research cigarettes, and observed that mainstream smoke from all the cigarettes contains substantial amounts of p-BSQ (100–200 μg/cigarette). We also show that when the CA fi lter is replaced by a charcoal fi lter, the amount of p-BSQ in the mainstream smoke is reduced by 73–80%, which is accompanied by a reduction of carbonyl formation in bovine serum albumin to the extent of 70– 90%. The charcoal fi lter also prevented cytotoxicity in A549 cells as evidenced by MTT assay, apoptosis as evidenced by FACS analysis, TUNEL assay, overexpression of Bax, activation of p53 and caspase 3, as well as emphysematous damage in a guinea pig model as seen by histology and morphometric analysis. The results indicate that the charcoal fi lter developed in our laboratory may protect smokers from cigarette smoke-induced cytotoxity, protein modifi cation, apoptosis and emphysema.

[Dey N, Das A, Ghosh A and Chatterjee I B 2010 Activated charcoal fi lter effectively reduces p-benzosemiquinone from the mainstream cigarette smoke and prevents emphysema; J. Biosci. 35 217–230] DOI 10.1007/s12038-010-0026-2

1. Introduction as free radicals, harmful gases, volatile organic compounds (VOCs), aldehydes, polycyclic aromatic hydrocarbons Cigarette is the world’s single most preventable (PAHs) and -specifi c nitrosamines (TSNA). Most cause of disease and . About one third of all adults in of these chemicals are considered to be causative agents the world are smokers (Slama 2008). Each year, over fi ve for CS-induced life-threatening diseases, particularly million people throughout the world die from smoking- cancer of the and other organs, myeloid leukaemia, related illness (IARC 2002). Cigarette smoke (CS) contains cardiovascular diseases and chronic obstructive pulmonary more than 4000 compounds (Stewart and Kleihues 2003). disease (COPD), including bronchitis and emphysema (Shah Among these, is the primary source of tobacco and Helfant 1988; Sherman 1991; Wald and Hackshaw dependence (US Department of and Human 1996; US Department of Health and Human Services Services 1988). Others are toxins and , such 1998; IARC 2002; World Cancer Report 2003; Harris et al.

Keywords. Apoptosis; charcoal fi lter; cigarette smoke; emphysema; p-benzosemiquinone Abbreviations used: AECS, aqueous extract of cigarette smoke; BSA, bovine serum albumin; CA-fi lter, cellulose acetate fi lter; CF, charcoal- fi ltered; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DI: destructive index; DMSO, dimethyl sulphoxide; ESR, electron spin resonance; H&E, haematoxylin–eosin; HPLC, high performance chromatography; HRP, horseradish peroxidase; Lm, mean linear intercept; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NMR, nuclear magnetic resonance; PAH, polycyclic aromatic hydrocarbon; PI, propidium iodide; PS, phosphatidyl serine; TLC, thin-layer chromatography; TSNA, tobacco-specifi c nitrosamines; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling; UV, ultraviolet; VOC, volatile organic compound http://www.ias.ac.in/jbiosci J. Biosci. 35(2), June 2010, 217–230, © Indian AcademyJ. Biosci. of 35 Sciences(2), June 2010 217 218 Neekkan Dey et al.

2004). Undoubtedly, the best way of preventing CS-induced we had demonstrated that p-BSQ largely mimics AECS in diseases is cessation of smoking. However, approaches to causing protein modifi cation, apoptosis and emphysematous cessation of smoking by public health campaigns and anti- lung damage (Banerjee et al. 2008). smoking laws passed by local governments have had limited A conventional CA fi lter is ineffective in adsorbing p- success. So, a practicable approach is to selectively reduce BSQ. We have also observed that two brands of commercial the toxins from mainstream smoke. One such approach charcoal-fi ltered cigarettes, Mild Seven Light and Magna, is the use of cigarette fi lters. Among the fi lters, cellulose containing uneven grain sizes of charcoal dispersed in CA acetate (CA) dominates the global fi lter market and charcoal tow, are ineffi cient in reducing p-BSQ from mainstream fi lters comprise about 10%. While a CA fi lter is effective CS. In fact, elimination of p-BSQ from the smoke depends in reducing only a little portion of the tar, it is not at all on the amount and the particular grain size of the activated selective for the other toxins of cigarette smoke. charcoal used (Chatterjee, US Patent 2006). Using human Charcoal-fi ltered (CF) cigarettes are predominantly lung epithelial cells (A549) as well as a guinea pig model, used (≈90%) in several countries, including Japan, Korea, here we show that an activated charcoal fi lter designed by us Venezuela and . It is less common (≈1%) in the (Chatterjee, US patent 2005; Chatterjee, Japan patent 2008; United States (Laugesen and Fowles 2006). However, Chatterjee, Korea patent 2008) effectively reduces p-BSQ these charcoal fi lter designs have little effect on the from mainstream CS and thereby prevents cytotoxicity, delivery of tar, nicotine and (Polzin et al. protein modifi cation, apoptosis and emphysematous lung 2008). Available data do not support the belief that use of damage in guinea pigs. commercial charcoal-fi ltered cigarettes reduces the risk of smoking-related diseases (Marugame et al. 2004; Muscat et 2. Materials and methods al. 2005; Laugesen and Fowles 2006). In particular, there do not appear be any studies on the effect of CF cigarettes on 2.1 Chemicals and reagents the prevention of COPD (Polzin et al. 2008; Marugame et al. 2004; Muscat et al. 2005; Coggins and Gaworski 2008; Activated charcoal (20–60 mesh) was purchased from Han-Jae et al. 2009). It is known that cigarette smoking is Sigma, USA. Granules of 60 mesh were separated by by far the commonest cause of COPD in western countries, grinding and sieving. An oxyblot protein oxidation detection accounting for about 95% of cases (Lopez and Murray 1998; kit was purchased from Intergen Company, USA. The in Pauwels and Rabe 2004; Barnes et al. 2003). Emphysema situ cell death detection kit was obtained from Roche, USA. is a prominent pathological feature of COPD. Emphysema The kit for protein estimation was obtained from Bio-Rad, is irreversible and currently there is no effective treatment USA. Antibodies against caspase 3, cleaved caspase 3, Bax, aimed at curing this fatal disease. This is particularly Bcl-2, p53, phospho-p53, anti-rabbit horseradish peroxidase because CS is a highly complex mixture of several thousand (HRP)-conjugate and anti-mouse HRP-conjugate as well compounds and it is not yet known whether a particular as the chemiluminescent kit for immunoblot analysis compound or a number of compounds are responsible for were obtained from Cell Signaling Technology, USA. The causing CS-induced emphysema. Once identifi ed, removal Annexin V-FITC kit was purchased from BD Biosciences. of that chemical entity could effectively reduce smokers’ Anti-tubulin antibody was obtained from Santa Cruz risk. Earlier, it was indicated that long-lived semiquinone Biotechnology, Inc, USA. All other chemicals were of radical(s) present in an aqueous extract of CS (AECS) is analytical grade. cytotoxic and causes protein and DNA damage (Pryor et al. 1983, 1986, 1998; Panda et al. 1999, 2000, 2001; Chouchane et al. 2006; Banerjee et al. 2008). Protein damage and DNA 2.2 Cigarettes fragmentation are hallmarks of emphysema (Tuder et al. 2003). We have isolated a major semiquinone from AECS Different brands of cigarettes were purchased from the and characterized it as p-benzosemiquinone by various local market and used without delay. All the cigarettes used, physicochemical analyses, including ultraviolet (UV), except Mild Seven Light (Japanese) and Magna (Russian), mass, nuclear magnetic resonance (NMR) and electron were conventional CA fi lter-tipped. Mild Seven Light and spin resonance (ESR) spectroscopy (Banerjee et al. 2008; Magna were charcoal fi lter cigarettes. Our fi nding may have Chatterjee, US patent 2005; Chatterjee, Japan patent 2008; limitations, because manufacturers might have changed a Chatterjee, Korea patent 2008; Chatterjee, Europe patent brand’s design or tobacco and consequent emissions. 2008). is an Indian commercial cigarette. CA fi lter- p-Benzosemiquinone (p-BSQ) is present in the tipped Kentucky reference cigarettes were obtained from mainstream smoke of all cigarettes, irrespective of the the University of Kentucky Tobacco and Health Research brands examined. Using various in vitro and in vivo analyses, Institute (Lexington, Kentucky).

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 219

2.3 Preparation of charcoal-fi ltered cigarettes p-BSQ present in the smoke was calculated from a standard curve obtained with pure p-BSQ. Activated charcoal fi lter cigarettes used in the present study were prepared from the original branded cigarettes 2.7 Measurement of protein damage by replacing the conventional CA fi lter with our activated charcoal fi lter using 150 mg charcoal granules of 60 mesh Protein damage was measured by carbonyl formation in (250 microns) (Chatterjee, US Patent 2006). Essentially, bovine serum albumin (BSA) after reaction with 2, 4- the charcoal fi lter we developed is a cavity fi lter in which dinitrophenyl hydrazine, similar to that done earlier in the activated charcoal granules are placed in a void space our laboratory (Panda et al. 1999). The incubation system between two segments of CA fi lters. One portion of the CA contained 1 mg BSA and 50 μl of smoke solution obtained fi lter (≈14 mm) is the mouthpiece and other portion (≈3 from cigarettes with or without a charcoal fi lter in a fi nal mm) constitutes a barrier between the charcoal bed and the volume of 200 μl of 50 mM potassium phosphate buffer, tobacco portion. The CA mouthpiece was used to prevent pH 7.4. After incubation for 1 h at 37°C, the protein was any leakage of charcoal granules in the mainstream smoke. precipitated with 200 μl of trichloroacetic acid solution and The various parts, i.e. the CA mouth piece, charcoal bed, thin the rest of the procedure followed was as described earlier CA fi lter placed in between the charcoal bed and the tobacco (Panda et al. 1999). The values are expressed as nmoles of portion as well as the tobacco portion, were all constructed carbonyl formed per mg BSA. in one single unit without any ventilation.

2.8 Measurement of nicotine 2.4 Preparation of aqueous extract of cigarette smoke (AECS) solution Smoke from a lit cigarette was allowed to dissolve in 2 ml of 50 mM potassium phosphate buffer, pH 7.4 and fi ltered Smoke from one cigarette was extracted with 1 ml of 50 mM through a 0.22 μm Millipore fi lter. One millilitre of the potassium phosphate buffer, pH 7.4, fi ltered through a 0.22 yellow coloured fi ltrate was extracted with 1 ml of methylene μm Millipore fi lter and the pH adjusted to 7.4, as described chloride by vigorous vortexing to extract the nicotine in the before in detail (Panda et al. 1999). The AECS thus obtained methylene chloride layer. Of the methylene chloride layer was used immediately. containing the nicotine, 500 μl was then vortexed with 500 μl of 50 mM HCl solution and the nicotine in the aqueous layer 2.5 Isolation and characterization of p-benzosemiquinone was estimated by HPLC analysis at 254 nm (Chatterjee, US (p-BSQ) patent 2005). About 5–10 μl of the aqueous layer was diluted to 200 μl with the mobile solvent, and 20 μl of this diluted p-BSQ was isolated from AECS by differential solvent solution was injected into the HPLC column. A standard extraction, thin-layer chromatography (TLC) and high solution of nicotine was prepared in a similar manner and performance liquid chromatography (HPLC) as described analysed. The parameters used were: Instrument, Shimadzu earlier (Banerjee et al. 2008). p-BSQ was characterized 10A; Column, Lichrospher® 100 RP-18 endcapped (5 by various physicochemical analyses, including UV, mass, μm), Merck; mobile solvent: 50 mM KH2PO4 solution: NMR and ESR spectroscopy as reported earlier (Banerjee accetonitrile:methanol (78:17: 5, v/v) containing 1 mM et al. 2008). sodium hepatane sulphonate, pH 5.0; fl ow rate: 0.3 ml /min. The retention time of nicotine was 4.185 min. The minimum amount of nicotine that could be detected by the HPLC 2.6 Measurement of the comparative yields of p-BSQ in analysis under these conditions was 10 ng. smoke solution prepared from different cigarettes p-BSQ in smoke solution was quantitatively measured by 2.9 Measurement of tar HPLC as described earlier (Banerjee et al. 2008). About 5–10 μl of the smoke solution, fi ltered through a 0.22 μm Tar was collected from the mainstream smoke by suction (30 Millipore fi lter, was diluted about 40 times with mobile cm water) through a Millipore fi lter unit. The Millipore fi lter solvent and 20 μl of this diluted solution was injected into (0.22 μm) was changed every 2 min to avoid clogging of the the HPLC (Simadzu 10A) with a UV detector set at 294 fi lter. For each cigarette, 4 fi lters were used. After complete nm using a normal phase silica column (Lichrospher ® burning of the tobacco, the fi lters were dried in a vacuum Si60, Merck). The mobile solvent was methylene chloride: desiccator and weighed. The difference in weight of the methanol (90:10, v/v) with a fl ow rate of 0.5 ml/min. fi lters before and after collecting the particulate portion was The retention time of p-BSQ was 8.808 min. The amount of the weight of the tar (Chatterjee, US patent 2005).

J. Biosci. 35(2), June 2010 220 Neekkan Dey et al.

2.10 Cell culture according to the manufacturer’s instruction. The stained cells were counted under a fl uorescence microscope (Olympus A549 cells were grown to 50–60% confl uence in HamF12 B). Nuclei were simultaneously counted by counterstaining medium containing 10% foetal calf serum (GIBCO-BRL, with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) USA), 100 units /ml penicillin, 100 μg/ml streptomycin (Santa Cruz Biotechnology, USA). The percentage of and 4 mM glutamine/ml. The cells were grown at 37°C in a TUNEL-positive cells was calculated (Banerjee et al. 2008). humifi ed incubator maintained in an atmosphere of 95% air and 5% CO . 2 2.14 Exposure of guinea pigs to cigarette smoke (CS)

2.11 Cytotoxicity assay The procedure we followed was essentially similar to that described earlier in detail (Banerjee et al. 2007, 2008). Male The cytotoxicity of the aqueous extract of mainstream smoke short-hair guinea pigs weighing 400–500 g were used for from CA-fi ltered and charcoal-fi ltered (CF) cigarettes was all the experiments. All animal treatment procedures met evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl the Institutional Animal Ethics Committee guidelines. The tetrazolium bromide (MTT) assay (Mosmann 1983). After guinea pigs were fed a vitamin C-free diet for 7 days to respective treatments with 50 μl/ml of the CA or CF AECS minimize the vitamin C level of tissues (Panda et al. 2000). solution for 1 h, the culture medium was replaced by medium This is because vitamin C is a potential inhibitor of CS- containing 0.5 mg/ml MTT and incubated for an additional induced protein oxidation (Panda et al. 1999, 2000), which 3 h. The blue MTT formazan was dissolved in 1 ml of would otherwise counteract the damaging effect of CS. The dimethyl sulphoxide (DMSO), and the absorbance values vitamin C-free diet given to the guinea pigs was similar to that were determined at 560 nm in a UV-VIS spectrophotometer described earlier (Misra et al. 2003). After 7 days of vitamin (Shimadzu UV-2540). C deprivation, the guinea pigs were subjected to CS exposure (either from CA-fi ltered or CF cigarettes) from 5 cigarettes (2 puffs per cigarette)/animal/day in a smoke chamber 2.12 Differentiation between apoptosis and necrosis by (Banerjee et al. 2007), along with oral supplementation of 1 FACS mg vitamin C/animal/day for 2 weeks (6 days a week, Sunday excluded). Deprivation of vitamin C was discontinued to In the early stages of apoptosis, changes occur in the avoid the onset of scurvy. One mg of vitamin C per day is plasma membrane. One of the cell surface alterations is approximately the minimum dose needed to prevent scurvy the translocation of phosphatidyl serine (PS) from the inner in the guinea pig (Banerjee et al. 2007). With this dose of surface of the cell membrane to the outer layer. Annexin V, vitamin C, there was no symptom of onset of scurvy in any of a Ca2+-dependent phospholipid-binding protein with high the guinea pigs during the experimental period. The smoke affi nity for phosphatidyl serine, is a sensitive probe for PS chamber was similar to that of a vacuum desiccator with an and can thereby detect early apoptotic cells in a live cell open tube at the top and a side tube fi tted with a stopcock, as population. Propidium iodide (PI) stains only the necrotic described earlier (Banerjee et al. 2007, 2008). The volume cells. To distinguish between apoptosis and necrosis, A549 of the chamber was 2.5 l. The cigarette placed at the top was cells (3 x 106) treated with 50 μl of CA-fi ltered or CF AECS lit and CS was introduced into the chamber containing the solution for 12 h were stained by PI and Annexin V-FITC guinea pig by applying a mild suction of 4 cm water through (Becton Dickinson) according to manufacturer’s protocols the side tube for 5 s. Thereafter, the vacuum was turned off and analysed using the FACS Calibur-Cell software and the guinea pig was further exposed to the accumulated (Becton Dickinson). A total of 10 000 events were acquired. smoke for another 40 s. The total duration of exposure to The cells were properly gated and a dual parameter dot plot smoke from one puff was thus 45 s. Altogether, 2 puffs per of FL2H (X-axis; PI fl uorescence, linear scale) vs FL1-H (Y- cigarette were given, allowing the animal 1 min rest in a axis; FITC-fl uorescence, logarithmic scale) was recorded. smoke-free atmosphere to breathe air between each puff. The gap between one cigarette and the next was 1 h. Pair-fed 2.13 Terminal deoxynucleotidyl transferase-mediated de- sham controls were subjected to air exposure instead of CS oxyuridine triphosphate nick end labelling (TUNEL) assay under similar conditions. The guinea pigs were divided into the following A549 cells (2 x 106) treated with 50 μl of AECS were fi xed experimental groups (N = 6/group): (i) exposed to air, (ii) with 4% p- and permeabilized with titron X- exposed to CA-fi ltered CS, (iii) exposed to charcoal-fi ltered 100 (0.1%) in 0.1% Na-citrate. The cells were then washed CS. After exposure to air or CS for up to 15 days, both the with phosphate buffered saline and subjected to TUNEL sham controls and the CS-exposed guinea pigs were deprived assay using an in situ cell death detection kit (Roche, USA) of food overnight and sacrifi ced next day by diethyl ether

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 221 . The lungs were then excised immediately and mM EDTA [ethylenediaminetetraacetic acid], pH 8.0; 0.1% processed for analysis. Triton-X100; 0.01 mg/ml aprotinin; 0.005 mg/ml leupeptin; 0.4 mM phenylmethanesulphonyl fl uoride [PMSF]; and 4 mM NaVO ). Lysates were then centrifuged at 20 000 g for 2.15 Histology of lung section for measurement of 4 10 min to remove insoluble material. The supernatant (30–50 emphysematous lung damage μg protein) was resolved on 12% sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) gel. After Lung damage caused by exposure to CS was quantifi ed by electrophoresis, the proteins were electrotransferred to a measuring the mean linear intercept (Lm) and destructive PVDF membrane, blocked with 5% non-fat milk (Bio- index (DI). Lm represents the average size of alveoli, Rad), and probed with antibodies against caspase 3, cleaved indicating the air space, which is increased in emphysema. caspase 3, Bax, Bcl-2, p53 and phospho-p53 (1:1000) for 1 h. It was measured by the technique originally described Thereafter, the blot was washed, exposed to HRP-conjugated by Dunnill (1962). Images randomly selected from secondary antibodies for 1 h, and fi nally detected by haematoxylin–eosin (H&E) stained lung sections were chemiluminescence, as done earlier (Banerjee et al. 2008). captured in an Olympus B microscope at 20X magnifi cation and analysed using the Dewinter Biowizard 4.1 software. The image was resized to a fi nal magnifi cation of 70X. 2.17 Statistical analysis Briefl y, a cross-hair grid consisting of horizontal and vertical lines (1 cm apart) was laid over the digital image on the All values are expressed as mean ± SD. Statistical computer screen. The number of times each line crossed an signifi cance was carried out using one-way ANOVA. The P alveolar wall, both horizontally and vertically, was manually values were calculated using appropriate F-tests. Difference counted. Results are expressed by the formula: with P values <0.05 were considered signifi cant. Lm = L/(X×m), 3. Results where Lm = mean linear intercept; L= total length of the lines in mm; X = magnifi cation factor and m = sum of 3.1 Charcoal fi lter effectively reduces p-BSQ from main- intercepts (the points where the horizontal and vertical stream smoke of different brands of cigarettes and prevents lines independently intercepted the alveolar walls). Four protein carbonyl formation independent lung sections per animal were analysed. The degree of destruction of the alveolar walls was Table 1 shows that mainstream smoke from different brands quantifi ed by measuring the DI following the microscopic of CA-fi ltered commercial cigarettes as well as Kentucky manual point count method of Saetta et al. (1985) using the research cigarettes contain high amounts of p-BSQ. When Dewinter Biowizard 4.1 software, as stated above, except the CA fi lters are replaced by charcoal fi lters, the p-BSQ that the lines of the cross-hair grid were 2 cm apart. The content is reduced by 73–80%. Earlier, we had reported that spaces directly under the cross-hair points were counted as p-BSQ from CS is a major toxic component that is largely either normal (N) or destroyed (D). The DI was computed responsible for carbonyl formation in proteins (Banerjee from the formula: et al. 2008). Table 1 indicates that when the CA fi lter is DI = D/(D+N) ×100(%), replaced by a charcoal fi lter, carbonyl formation in BSA is markedly reduced. Table 1 further shows that tar and where D indicates destroyed and N indicates normal points. nicotine delivery are also considerably reduced in smoke An alveolar space was considered to be normal if it was from charcoal-fi ltered cigarettes. In a separate experiment, surrounded by intact walls or by wall disrupted in only one we have observed that fortifi cation of tobacco (1 g) with 3 place. Alveolar space was considered to be destroyed when mg nicotine per cigarette leads to increased nicotine delivery the wall of an alveolus was disrupted in two or more places. in the mainstream smoke to almost the original level (data not shown). However, such fortifi cation does not cause any 2.16 Western blot increase in the p-BSQ content of the smoke, apparently because nicotine is not a precursor of p-BSQ. For western blot, cells (3 x 106) were treated with CA- fi ltered or charcoal-fi ltered AECS solution for 1 h and then 3.2 Charcoal fi lter prevents AECS-induced alteration of reincubated in fresh Ham F12 medium for 16 h. Control cells morphology and loss of viability in A549 cells received no treatment. After that, cell extracts were prepared by lysing the cells in lysis buffer (20 mM Tris [tris(hydroxy In the pathogenesis of CS-induced pulmonary diseases, methyl)aminomethane chloride], pH 7.4; 250 mM NaCl; 2 injury of the alveolar epithelium is an important process.

J. Biosci. 35(2), June 2010 222 Neekkan Dey et al.

Table 1. p-Benzosemiquinone (p-BSQ), bovine serum albumin (BSA) oxidation, nicotine delivery and tar content in aqueous extract of cigarette smoke (AECS) prepared from different international cigarettes with and without an activated charcoal fi lter. The amount of charcoal used in the fi lter was 150 mg of grain size 60 mesh. S. no. Condition with Brand of p-BSQ† Per cent BSA* oxidation Nicotine‡ Tar¶ content or without cigarette content (μg) reduction in (nmoles of delivery (mg) (mg) charcoal fi lter p-BSQ carbonyl content** formed)# 1 Without Kentucky 100 ± 7.07 -- 6.0 0.75 9 With 3R4F† 20 ± 1.79 80 1.5 0.40 4 2 Without Kentucky 180 ± 17.90 -- 10.0 1.16 15 With 1R3F† 40 ± 2.83 78 3.0 0.50 10 3 Without Wills 200 ± 17.90 -- 10.0 1.00 20 With Navy Cut§ 40 ± 3.16 80 2.5 0.45 12 4 Without Winston 200 ± 20.00 -- 12.4 1.3 18 With 45 ± 4.47 77 3.0 0.55 11 5 Without 180 ± 16.10 -- 10.6 1.3 17 With 45 ± 8.94 75 2.8 0.52 10 6 Without Viceroy 175 ± 13.40 -- 10.4 1.3 16 With 40 ± 4.56 73 2.7 0.54 9 7 Without Marlboro 170 ± 13.40 -- 10.0 1.2 16 With 36 ± 3.44 79 2.5 0.50 10 8 Without Benson & 170 ± 14.30 -- 10.2 1.1 16 With Hedges 34 ± 3.58 80 2.5 0.47 9 9 Without Virginia 160 ± 14.30 -- 9.8 1.1 14 With Slims 34 ± 4.47 79 2.4 0.46 8 10 Without Cambridge 160 ± 13.40 -- 10.0 1.0 15 With 36 ± 4.50 77 2.6 0.44 9 11 Without Kent 150 ± 9.80 -- 8.5 1.0 14 With 30 ± 2.83 80 2.2 0.46 9 12 Without Kool 155 ± 11.40 -- 9.0 1.0 14 With 32 ± 4.56 79 2.0 0.44 9 13 Without Classic 135 ± 8.94 -- 7.5 0.8 12 With 25 ± 3.69 79 1.6 0.40 8 14 Without Monte Carlo 140 ± 9.88 -- 7.0 0.8 12 With 25 ± 2.68 78 1.5 0.40 8 15 Without Mild 100 ± 7.07 -- 6.0 0.7 8 With Seven Light* 20 ± 2.28 80 1.4 0.38 3 16 Without Magna* 110 ± 8.10 -- 6.5 0.6 9 With 22 ± 2.83 80 1.5 0.36 4 All cigarettes are with original fi lters provided by the manufacturers; †Kentucky Research Cigarettes; §Indian commercial cigarette; *Mild Seven Light (Japanese) and Magna (Russian) are charcoal fi lter cigarettes; #Amount of carbonyl formed in 1 mg BSA using 50 μl of aqueous extract of CS. Details of the incubation system and measurements of p-BSQ, carbonyl, nicotine and tar are given in Materials and methods. ** Values are means of six independent determinations ± SD.

We compared the effects of CA-fi ltered AECS with cells were exposed to charcoal-fi ltered AECS, both the charcoal-fi ltered AECS on the morphology and viability of confl uence and the morphology were almost similar to that A549 cells. Examination under a phase-contrast microscope of control cells (fi gure 1A, fi rst and third panels). Similar showed that exposure of the cells to CA-fi ltered AECS observations were made regarding the cytotoxicity of AECS. caused an increase in fl oating cells accompanied by a MTT assay revealed that compared with the normal controls, decrease in the density of cells. The cells still attached CA-fi ltered AECS reduced the viability of the cells to about became round in shape and the gap between the cells was 32%. In contrast to this, the viability of charcoal-fi ltered enlarged (fi gure 1A, middle panel). However, when the AECS-treated cells was about 78% (fi gure 1B).

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 223

Figure 1. Effect of charcoal fi lter on aqueous extract of cigarette smoke (AECS)-induced morphology and viability of A549 cells. (A) Phase-contrast micrographs of control (without treatment); cellulose acetate (CA)-AECS, after 1 h treatment with CA-fi ltered AECS solution and charcoal-fi ltered AECS, after 1 h treatment with charcoal-fi ltered AECS solution. Photographs were taken 12 h after the respective treatments as signifi cant morphological changes were observed during this period. (B), Bar diagram showing the percentage of viable cells compared with the control as determined by MTT cell viability assay 24 h after the respective treatments. Data are expressed as the mean ± SD; N=6.

3.3 Charcoal fi lter prevents AECS solution-induced apop- control cells (1.49%; P<0.05). This indicates that the CA- tosis in A549 cells as evidenced by FACS analysis using fi ltered AECS-treated cells were entering into the apoptotic Annexin V and PI phase. However, when the cells were treated with 50 μl/ml medium charcoal-fi ltered AECS solution, the percentage of Flowcytometric data (fi gure 2) indicated that when A549 Annexin V-positive cells was only 2.18%, indicating that the cells were treated with 50 μl/ml medium of CA-fi ltered charcoal fi lter prevented AECS solution-induced apoptosis AECS solution there was a signifi cant increase (78.92%) in A549 cells. In all the cases, the percentage of PI-positive of Annexin V-positive cells compared with the untreated necrotic cells were negligible.

J. Biosci. 35(2), June 2010 224 Neekkan Dey et al.

3.5 Charcoal fi lter prevents AECS-induced apoptosis in A549 lung epithelial cells as evidenced by immunoblotting

Apart from the Annexin V-PI staining by FACS analysis and DNA fragmentation (TUNEL assay), apoptosis was evidenced in A549 cells by activation of p53, increase in Bax proteins and activation of caspase 3 (fi gure 4). The level of p53 remained unaltered in all the groups irrespective of the treatment they received. However, fi gure 4A (panel I, lanes 4 and 5) shows that the level of phosphorylated p53 (active form) markedly increased when A549 cells were exposed to CA-fi ltered AECS (30 μl/ml or 50 μl/ml medium). There was no activation of p53 in the case of either untreated control (lane 1) or cells treated with charcoal-fi ltered AECS (30 μl/ml or 50 μl/ml medium,.lanes 2 and 3). It is known that one of the mechanisms of apoptosis is overexpression of Bax, a member of the Bcl-2 family. After treatment with CA-fi ltered AECS (30 μl/ml or 50 μl/ml medium), the level of Bax protein increased (fi gure 4A, panel II). In contrast to this, there was no overexpression of Bax in the case of the untreated control cells (lane 1) or cells treated with charcoal-fi ltered AECS (30 μl/ml or 50 μl/ml medium; lanes 2 and 3). It is known that while Bax is proapoptotic, Bcl-2 is anti-apoptotic. We therefore examined the level of Bcl-2 protein. While the level of Bax protein increased in response to CA-fi ltered AECS treatment, the level of Bcl-2 protein remained unaffected (fi gure 4A, panel III) in all the cases. These observations suggest that the apoptotic effect of Figure 2. Charcoal fi lter prevents aqueous extract of cigarette CA-fi ltered AECS on A549 cells is caused by an increase of smoke (AECS) solution-induced apoptosis in A549 cells as evidenced by FACS analysis using Annexin V and propidium activated p53 as well as an increased Bax/Bcl-2 ratio, which iodide (PI) double staining technique. After treatment with is completely prevented by the charcoal fi lter. cellulose acetate (CA)-fi ltered AECS solution and charcoal-fi ltered CA-fi ltered AECS-induced apoptosis was further AECS solution, A549 cells were labelled with PI and AnnexinV- supported by checking the level of cleaved caspase 3 by FITC and then analysed on a fl owcytometer. Controls were cells western blotting of A549 cell lysate using anti-caspase without treatment with AECS, but double-stained with PI and 3 antibody (fi gure 4B, panel I). The level of the cleaved AnnexinV-FITC. Details are given in the Materials and methods product of caspase 3 (17 kDa) was markedly increased section 2.12. Dual parameter dot plot of FITC fl uorescence (Y-axis) (fi gure 4B, panel II, lanes 4 and 5) in response to CA-fi ltered vs PI (X-axis) has been shown as fl uorescence intensity. Quadrants: AECS treatment (30 μl/ml or 50 μl/ml medium). There lower left, viable cells; upper left, apoptotic cells; upper right, late was no activation of caspase 3 in the case of the untreated apoptotic and lower right, necrotic cells. control cells (fi gure 4B, panel II, lane 1) or cells treated with charcoal-fi ltered AECS (30 μl/ml and 50 μl/ml medium, 3.4 Charcoal fi lter prevents AECS-induced DNA fragmen- respectively; lanes 2 and 3). tation of A549 cells as evidenced by TUNEL assay

When A549 cells were exposed to CA-fi ltered AECS (50 3.6 Smoke from charcoal-fi ltered cigarettes prevents μl/ml medium), the percentage of TUNEL-positive nuclei emphysema as evidenced by air space enlargement and markedly increased (90 ± 5 SD), as indicated by green parenchymal destruction fl uorescence attributable to fl uorescein-dUTP labelling (fi gure 3 A, B, upper panel). The lower panel shows the Histology profi les showed that when the guinea pigs are nuclei counterstained with DAPI. In contrast, there was exposed to CA-fi ltered CS for two weeks at an exposure rate little increase (4 ± 1 SD) in TUNEL-positive cells exposed of 5 cigarettes (2 puffs/cigarette)/guinea pig/day, there was to charcoal-fi ltered AECS (fi gure 3 A, B). This indicates marked emphysematous damage of the lung, as compared that AECS-induced oligonucleosomal fragmentation is with sham control guinea pigs exposed to air (fi gure 5 A, prevented by the charcoal fi lter. B). The damage was evidenced by morphometric change

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 225

Figure 3. (A) Charcoal fi lter prevents aqueous extract of cigarette smoke (AECS)-induced DNA fragmentation of A549 cells as evidenced by TUNEL assay. Cells were incubated with or without 50 μl/ml of cellulose acetate (CA)-fi ltered AECS or charcoal-fi ltered AECS. After 24 h, cells were analysed by terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling (TUNEL) staining for the presence of apoptotic bodies (as described in Materials and methods). Upper panel: cells were stained with fl uorescein-labelled dUTP according to the manufacturer’s protocol. Green fl uorescence indicates TUNEL-positive cells. Lower panel: cells were stained with DAPI to identify the cell nuclei. Six images were analysed in two different experimental sets (3 images/set) from each group, respectively; (B) quantitative evaluation of TUNEL-positive cells; the bars over the respective columns represent means ± SD; * indicates statistically signifi cant, P<0.001 with respect to the control. and enlargement of air spaces. In contrast, when the guinea pigs, both the Lm and DI (0.449 ± 0.045 and 58.2 ± 5.4, pigs were exposed to smoke from charcoal-fi ltered (CF) respectively) of CA-fi ltered CS-exposed guinea pigs are cigarettes, no apparent lesion in the lung cells was observed signifi cantly increased (P<0.05). In contrast, there is no and the histological profi le appeared to be similar to that of signifi cant difference between the Lm (0.390 ± 0.020) and normal guinea pigs (fi gure 5 C). Quantitative evaluation DI (38.8 ± 6.3) of the lung sections of guinea pigs exposed of lung damage was done by measuring the mean Lm and to charcoal-fi ltered CS and those of sham controls exposed DI of the infl ated lung sections (table 2). The number of to air (P>0.05). The results confi rm that CS-induced guinea pigs used in each group was eight. Four representative emphysema is signifi cantly prevented when the CA fi lter is non-overlapping images from each lung section replaced by a charcoal fi lter. were captured in an Olympus B microscope attached with a CCD Cool camera and analysed by the Dewinter 4. Discussion Biowizard 4.1 software. Sixteen images were analysed in each group. Table 2 shows that compared with the Emphysema is a prominent feature of COPD, which is Lm (0.379 ±0.009) and DI (35.3 ± 7.2) of normal guinea a major and increasing global cause of mortality and

J. Biosci. 35(2), June 2010 226 Neekkan Dey et al.

In this paper, we have made a comparative evaluation of the p-BSQ content, tar content, protein carbonyl formation, toxicity, apoptosis and the extent of emphysematous lung damage produced by CA-fi ltered cigarettes and the CF cigarettes developed in our laboratory. The CF cigarettes were prepared by replacing the CA fi lters with a charcoal fi lter. The weight of tobacco, tobacco blend and the smoking conditions remained essentially similar. Hence, the effect of the charcoal fi lter on the biological activity of mainstream smoke was comparable with that of the CA fi lter. We have shown that the charcoal fi lter is far superior to the CA fi lter in all aspects. Earlier, we had shown that p-BSQ of CA- fi ltered AECS is largely responsible for carbonyl formation in proteins (Banerjee et al. 2008). Here, we have presented data to indicate that a charcoal fi lter not only reduces about 73–80% of the p-BSQ from the mainstream smoke, but also prevents carbonyl formation in BSA to the extent of 70–79%, indicating a correlation between the reduction of p-BSQ content and carbonyl formation. A charcoal fi lter also reduces the tar content of CS to about 33–56% and nicotine content to the extent of 37–58%. However, the nicotine content of CS can be replenished almost to the original level Figure 4. (A) Immunoblot of phosphorylated p53, p53, Bax and by fortifi cation of the tobacco with nicotine. Fortifi cation by Bcl-2 in cell lysate of A549 cells treated with charcoal-fi ltered nicotine does not cause any increase in the p-BSQ content, and cellulose-acetate (CA)-fi ltered aqueous extract of cigarette apparently because nicotine is not a precursor of p-BSQ. smoke (AECS) solution. Lane 1, untreated control; lanes 2 and Nicotine is a pharmacological component and a primary 3, cells treated with 30 μl and 50 μl charcoal-fi ltered AECS/ml, source of tobacco dependence. The amount of nicotine respectively; lanes 4 and 5, cells treated with 30 μl and 50 μl present in the CS of a cigarette does not appear to be a cause CA-fi ltered AECS/ml, respectively. (B) Immunoblot of caspase 3 of CS-induced toxicity and apoptosis (Ramage et al. 2006). and cleaved caspase 3 in cell lysate of A549 cells treated with CF and CA-fi ltered AECS solution. Lane 1, untreated control; lanes It has been reported that the semiquinone of CS is 2 and 3, treated with 30 μl and 50 μl CF-AECS/ml, respectively; cytotoxic (Chouchane et al. 2006; Pryor et al. 1998). Here lanes 4 and 5, treated with 30 μl and 50 μl CA-fi ltered AECS/ml, we show that a charcoal fi lter, which effectively reduces the respectively. α-tubulin was used as the loading control. p-BSQ of mainstream smoke, also causes a marked reduction in the cytotoxicity of CS. Cytotoxicity was determined by morbidity (Lopez and Murray 1998; Pauwels and Rabe microscopic examination of cell morphology and viability 2004). Cigarette smoking is by far the commonest cause of by MTT assay. emphysema in western countries, accounting for about 95% Previous observations from our laboratory had indicated of cases (Barnes et al. 2003). The cellular and molecular that the initial event of exposure of lung cells to CA-fi ltered mechanisms of CS-induced emphysema remain unclear, CS is protein damage, which is followed by apoptosis particularly because CS contains about 4000 compounds (Banerjee et al. 2008). Once protein damage is prevented, (Stewart and Kleihues 2003). Earlier, we had isolated p-BSQ, apoptosis is also prevented (Banerjee et al. 2008). Earlier, a long-lived radical from an Indian commercial CA-fi ltered we had demonstrated that p-BSQ largely mimics AECS- CS, and demonstrated that p-BSQ largely mimics CS- induced apoptosis. Here we show that reduction of p-BSQ induced protein modifi cation, apoptosis and emphysematous from mainstream smoke by a charcoal fi lter is accompanied lung damage in a guinea pig model (Banerjee et al. 2008). In by prevention of apoptosis. Apoptosis is an important mode this study, we have shown that besides the Indian cigarette, of cell death under both physiological and pathophysiological substantial amounts of p-BSQ are present in the mainstream conditions. Several techniques are available for the study smoke of all the CA-fi ltered cigarettes studied, irrespective and quantitation of apoptosis in cell culture. Two commonly of the brand. We also examined Kentucky reference used techniques to quantify apoptosis are: (i) FACS research cigarettes, whose tobacco blend, nicotine content analysis using Annexin V and (ii) measurement of DNA and toxicity have been reported to be representative of other fragmentation using the TUNEL method (Rao et al. 1998; cigarettes (Han-Jae et al. 2009; Doolittle et al. 1990; Chen Whiteside et al. 1998; Whiteside and Munglani 1998). Here, and Moldoveanu 2003). we show using both the techniques that apoptosis produced

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 227

Figure 5. Histology of lung sections of guinea pigs exposed to cellulose-acetate (CA)-fi ltered cigarette smoke and charcoal-fi ltered cigarette smoke stained with haematoxylin and eosin. Control, guinea pigs exposed to air; CA-fi ltered CS, guinea pigs exposed to CA- fi ltered cigarette smoke for 14 days; charcoal-fi ltered CS, guinea pigs exposed to charcoal-fi ltered cigarette smoke for 14 days. Details of exposure of guinea pigs are given in Materials and methods. Data on quantitative evaluation of morphometry, as measured by mean linear intercept (Lm) and destructive index (DI), are given in table 1.

Table 2. Measurements of mean linear intercept (Lm) and contribute to apoptosis through disassembly of cell structures destructive index (DI) of lung sections of normal and cigarette by disrupting the nuclear structure and cleaving several smoke-exposed guinea pigs cytoskeletal proteins (Schraufstatter et al. 1984; Thornberry Condition of exposure Mean linear Destructive index and Lazebnik 1998). Caspases, synthesized initially as intercept (Lm) (DI) Mean ± SD inactive single polypeptide chains, undergo proteolytic Mean ± SD cleavage to produce subunits (cleaved caspase 3, e.g. 17 kDa) Air (normal) 0.379 ± 0.009 35.3 ± 7.2 having protease activity. We have shown that while cleaved Smoke from cellulose 0.449 ± 0.045 58.2 ± 5.4 caspase 3 is produced by using a CA fi lter, it is not produced acetate-fi ltered when the CA fi lter is replaced by a charcoal fi lter. cigarettes§ Apoptosis is regulated by the expression of Bax, a Smoke from charcoal- 0.390 ± 0.020 38.8 ± 6.3 member of the Bcl-2 protein family (Kluck et al. 1997; fi ltered cigarettes§ Tsujimoto 1998). Bax is pro-apoptotic and Bcl-2 is anti- apoptotic. Here, we show that exposure of A549 cells to CA- §Wills Navy Cut (Indian commercial cigarette) The number of fi ltered AECS results in overexpression of Bax, but no such guinea pigs used in each group was 8. The animals were exposed to air or smoke for two weeks (6 days a week). Several 5 μm overexpression takes place when the CA fi lter is replaced by sections from the middle lobe of both the left and right lungs a charcoal fi lter. The level of Bcl-2 protein remains unaltered were stained with haematoxylin and eosin. Four representative in both the cases. non-overlapping images from each section were captured in an It is known that the phosphorylated form of p53 Olympus B microscope attached with a CCD Cool camera and increases in response to DNA damage (Banin et al. 1998). analysed by the Dewinter Biowizard 4.1 software. Altogether Using TUNEL assay, we have shown that marked DNA 16 images (approximately 1600 point counts) were analysed in fragmentation occurs by exposure of A549 cells to CA- each group. Details are given in Materials and methods. P values fi ltered AECS. We have further shown that such exposure between cellulose acetate-fi ltered smoke exposed and normal (air also causes an increase in phospho-p53. No such increase exposed, normal) are: for Lm, P = 0.0039; DI, P = 0.0001 (highly in phospho-p53 occurred when the cells were exposed to signifi cant) and that between charcoal-fi ltered smoke exposed charcoal-fi ltered AECS. and normal are: for Lm, P = 0.27; DI = 0.80 (not signifi cant). Previously, we had shown that exposure of guinea pigs to smoke from an Indian CA-fi ltered cigarette in A549 cells by AECS prepared from CA-fi ltered cigarette causes emphysematous lung damage (Banerjee et al. smoke is prevented when the CA fi lter is replaced by a 2007, 2008). Emphysema is defi ned as the ‘abnormal charcoal fi lter. permanent enlargement of the airspaces distal to the Caspases are aspartate-directed cysteine proteases with a terminal bronchioles, accompanied by destruction of their pivotal role in apoptosis. Caspase 3 is an important caspase walls’ (American Thoracic Society 1995). The validity in the execution of downstream events in apoptosis. Caspases of a potential animal model of emphysema is tested by

J. Biosci. 35(2), June 2010 228 Neekkan Dey et al. quantitative histopathological methods measuring both studies. Until now, limited epidemiological data are airspace enlargement and destruction of the alveolar walls. available to demonstrate a conclusive benefi cial effect of Measurements of Lm and DI are authentic parameters for commercial CF cigarettes (Coggins and Gaworski 2008). In quantitation of enlargement of airspaces and destruction our study, all the guinea pigs exposed to CA-fi ltered CS had of the alveolar walls (Robbesom et al. 2003; Saetta et al. emphysematous lesions, whereas only about 15% of smokers 1985; Fehrenbach 2006). We have shown that both the Lm develop emphysema (Snider et al. 1985). So, some genetic and DI of lung sections produced by exposure of guinea predisposition and nutritional status of vitamin C might be pigs to smoke from a CA-fi ltered cigarette are signifi cantly involved in the susceptibility of a smoker to emphysema. increased. However, when the CA fi lter is replaced by a Nevertheless, since there is no curative therapy available charcoal fi lter, the values of Lm and DI are comparable with for emphysema, a practical approach would be prevention. those of normal sham control guinea pigs, indicating that If the results obtained with guinea pigs are applicable to a charcoal fi lter prevents CS-induced emphysematous lung humans, use of a charcoal fi lter developed in our laboratory damage in guinea pigs. may protect smokers from emphysema. A number of reports from other laboratories indicate that induction of emphysematous lesions in guinea Acknowledgements pigs by CS requires exposure times of at least several months (Wright and Churg 1995, 2002; Wright et al. 2002). The authors acknowledge fi nancial support from Council of In contrast to this, we produced emphysema within 14 Scientifi c and Industrial Research, New Delhi, for carrying days of CS exposure. The apparent difference is because out this research. ND is a Juthika Research Fellow; AD is we used vitamin C-restricted guinea pigs to minimize a Phulrenu Guha Research Fellow; AG is an ICMR Senior the vitamin C level in the tissues (Banerjee et al. 2007). Research Fellow and IBC is an INSA Honorary Scientist. Vitamin C is a potential inhibitor of CS-induced protein damage (Panda et al. 1999), which is apparently an initial event in the development of emphysema (Banerjee et al. References 2008). In conclusion, we report that irrespective of the brand American Thoracic Society 1995 Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease; of CA-fi ltered cigarettes examined, the mainstream smoke Am. J. Respir. Crit. Care Med. 152 S77–S121 contains p-BSQ, a major, highly toxic, long-lived water Banerjee S, Maity P, Mukherjee S, Sil A K, Panda K, Chattopadhyay soluble radical. The amount of p-BSQ varies with the D J and Chatterjee I B 2007 Black tea prevents cigarette smoke- tar content. We have examined 14 brands of CA-fi ltered induced apoptosis and lung damage; J. Infl ammation 4 3 cigarettes from different countries of the world, including Banerjee S, Chattopadhyay R, Ghosh A, Koley H, Panda K, Roy India, England, USA, as well as Kentucky Research S, Chattopadhay D J and Chatterjee I B 2008 Cellular and Cigarettes and 2 brands of CF-fi ltered cigarettes from molecular mechanisms of cigarette smoke-induced lung damage Japan and and observed that smoke from all the and prevention by vitamin C; J. Infl ammation 5 21 cigarettes contains substantial amounts of p-BSQ (100–200 Banin S, Moyal L, Shieh S, Taya Y, Anderson C W, Chessa μg/cigarette). This indicates that p-BSQ is a prominent toxic L, Smorodinsky N I, Prives C et al. 1998 Enhanced phosphorylation of p53 by ATM in response to DNA damage; component of CS, irrespective of the source of the cigarette. Science 281 1674–1677 Previous reports from our laboratory in human lung epithelial Barnes P J, Shapiro S D and Pauwels R A 2003 Chronic obstructive cells (A549) and in vivo in guinea pigs indicate that p-BSQ pulmonary disease: molecular and cellular mechanisms; Eur. from CA-fi ltered CS causes protein damage, apoptosis and Respir. J. 22 672–688 emphysematous lung lesions (Banerjee et al. 2008). Here we Chatterjee I B 2005 Process for the isolation of a major harmful show that when the CA fi lter is replaced by a charcoal fi lter oxidant from cigarette smoke (US Patent No. 6,929,012) developed in our laboratory, p-BSQ is markedly reduced Chaterjee I B 2008 Process for the isolation of a major harmful from the mainstream smoke and all these pathophysiological oxidant from cigarette smoke (Korea Patent No. 10-0868687) events are prevented. Chatterjee I B 2008 Process for the isolation of a major harmful Apparently, the results obtained with guinea pigs may be oxidant from cigarette smoke (Japan Patent No. 4094545) Chatterjee I B 2006 Activated charcoal fi lter for effectively extrapolated to human smokers. The structure of the guinea reducing p-benzosemiquinone from the mainstream cigarette pig lung is similar to that of human lung (Wright and Churg smoke (US Patent No. 7,025,067 B2)Chatterjee I B 2008 Japan 2002). In addition, the guinea pig develops morphological Patent No. 3966856 Activated charcoal fi lter for effectively and pathophysiological alterations after exposure to CS in the reducing p-benzosemiquinone from the mainstream cigarette same pattern as humans (Wright and Churg 2002). However, smoke the present study has some limitations. The results obtained Chatterjee I B 2008 Europe Patent No. EP1434503 (validated in UK, with guinea pigs should be validated by epidemiological Italy, , Spain, Germany, Portugal and ) Activated

J. Biosci. 35(2), June 2010 Activated charcoal fi lter prevents emphysema 229

charcoal fi lter for effectively reducing p-benzosemiquinone and is exclusively caused by its tar phase: prevention from the mainstream cigarette smoke by vitamin C; Toxicol. Lett. 123 21–23 Chen P X and Moldoveanu S C 2003 Mainstream smoke chemical Panda K, Chattopadhyay R, Ghosh M K, Chattopadhyay D J and analysis for 2R4F Kentucky reference cigarette; Beitr. Chatterjee I B 1999 Tabakforsch. 20 448–458 Vitamin C prevents cigarette smoke induced oxidative damage of Chouchane S, Wooten J B, Tewes F J, Wittig A, Müller B P, Veltel proteins and increased proteolysis; Free Radic. Biol. Med. 27 D and Diekmann J 2006 Involvement of semiquinone radicals in 1064–1079 the in vitro cytotoxicity of cigarette mainstream smoke; Chem. Panda K, Chattopadhyay R, Ghosh M K, Chattopadhyay D J and Res. Toxicol. 19 1602–1610 Chatterjee I B 2000 Coggins C R E and Gaworski C L 2008 Could charcoal fi ltration Vitamin C prevents cigarette smoke induced oxidative damage in of cigarette smoke reduce smoking-induced diseases? A review vivo; Free Radic. Biol. Med. 29 115–124 of the literature; Regulatory and Pharmacology 50 Pauwels R A and Rabe K F 2004 Burden and clinical features of 359–365 chronic obstructive pulmonary disease (COPD); Lancet 364 Doolittle D J, Lee C K, Ivett J L, Mirsalis J C, Ricco E, Rudd C 613–620 J, Burger G T and Hayes A W 1990 Comparative studies on Pryor W A, Deoley M M and Church D F 1986 The inactivation of the genotoxic activity of mainstream smoke condensate from α1-proteinase inhibitor by gas-phase cigarette smoke: protection cigarettes which burn or only heat tobacco; Environ. Mol. by antioxidants and reducing species; Chem. Biol. Interact. 57 Mutagen. 15 93–105 271–283 Dunnill M S 1962 Quantitative methods in the study of pulmonary Pryor W A, Prier D G and Church D F 1983 Electron spin resonance pathology; Thorax 17 320–328 study of mainstream and side stream cigarette smoke: nature of Fehrenbach H 2006 Animal models of pulmonary emphysema: a the free radicals in gas phase smoke and cigarette tar; Environ. stereologist’s perspective; Eur. Resp. Rev. 15 136–147 Health Perspect. 47 345–355 Han-Jae S, Hyung-Ok S, Jung-Ho H, Chul-Hoon P, Hyeong-Seok Pryor W A, Stone K, Zang L Y and Bermudez E 1998 Fractionation L, Dong-Wook L, Keon-Joong H and Hak-Chul H 2009 Effect of cigarette tar extracts: fractions that contain the tar radical of cigarette fi lters on the chemical composition and in vitro cause DNA damage; Chem. Res. Toxicol. 11 441–448 biological activity of cigarette mainstream smoke; Food Chem. Polzin G M, Zhang L, Hearn B A, Tavakoli A D, Vaughan C, Ding Toxicol. 7 192–197 Y S, Ashley D L and Watson C H 2008 Effect of charcoal- Harris J E, Thun M J, Mondul M L and Calle E E 2004 Cigarette containing cigarette fi lters on gas phase volatile inorganic tar yields in relation to mortality from in the caner compounds in mainstream cigarette smoke; Tob. Control 17 prevention study II prospective cohort, 1982–8; BMJ 328 i10–i16 72–76 Ramage L, Jones A C and Whelan C J 2006 Induction of apoptosis International Agency for Research on Cancer 2002 IARC with and related products in A549 lung epithelial monographs on the evaluation of the carcinogenic risk of cells in vitro; J. Infl amm. 3 3 chemicals to humans (Lyon, France: IARC) p. 83 Rao J K, Letada P, Haverstick D M, Herman M M and Savory J Kluck R M, Bosy-Wetzel E, Green D R and Newmeyer D D 1997 1998 Modifi cations to the in situ TUNEL method for detection The release of cytochrome c from mytochondria: a primary site of apoptosis in paraffi n-embedded tissue sections; Ann. Clin. for Bcl-2 regulation of apoptosis; Science 276 1132–1136 Lab Sci. 28 131–137 Laugesen M and Fowles J 2006 Marlboro UltraSmooth: a Robbesom A A, Versteeg E M M, Veercamp J H, van Krieken J H potentially reduced exposure cigarette?; Tob. Control 15 J M, Bulten H J, Smits H T J, Willems L N A, van Herwaarden 430–435 C L A et al. 2003 Morphological quantifi cation of emphysema Lopez A D and Murray C C 1998 The global burden of disease, in small human lung specimens: comparison of methods and 1990–2020; Nat. Med. 4 1241–1243 relation with clinical data; Mod. Pathol. 16 1–7 Marugame T Sobue T, Nakayama T Suzuki T, Kuniyoshi H, Saetta M, Shiner R J, Angus G E, Kim W D, Wang N, King Sunagawa K, Genka K, Nishizawa N et al. 2004 Filter cigarette M, Ghezzo H and Cosio M G 1985 Destructive index: a smoking and lung cancer risk; a hospital-based case–control measurement of lung parenchymal destruction in smokers; Am. study in Japan; Br. J. Cancer 90 646–651 Rev. Respir. Dis. 131 764–769 Misra A, Chattopadhyay R, Banerjee S, Chattopadhyay D J and Schraufstatter I U, Revak S D and Cochrane C G 1984 Protease Chatterjee I B 2003 Black tea prevents cigarette smoke-induced and oxidants in experimental pulmonary infl ammatory injury; J. oxidative damage of proteins in guinea pigs; J. Nutr.133 Clin. Invest. 73 1175–1184 2622–2628 Sherman C B 1991 Health effect of cigarette smoking; Clin. Chest Mosmann T 1983 Rapid colorimetric assay for cellular growth and Med. 12 643 survival: application to proliferation and cytotoxicity assays; J. Shah P K and Helfant R H 1988 Smoking and coronary artery Immunol. Methods 65 55–63 diseases; Chest 94 449–452 Muscat J E, Takezaki T, Tajima K and Stellman SD 2005 Charcoal Slama K 2008 Global perspective on . Part I. The cigarette fi lters and lung cancer risk in Aichi Prefecture; Japan global state of the tobacco epidemic; Int. J. Tuberc. Lung Dis. Cancer Sci. 96 283–287 12 3–7 Panda K, Chattopadhyay R, Ghosh M K, Chattopadhyay D J and Snider G L, Lucey E C and Stone P J 1985 Animal models of Chatterjee I B 2001 Cigarette smoke-induced protein oxidation emphysema; Am. Rev. Respir. Dis. 133 149–169

J. Biosci. 35(2), June 2010 230 Neekkan Dey et al.

Stewart B W and Kleihues P (eds) 2003 World cancer report 2003 Wald N J and Hackshaw A K 1996 Cigarette smoking: an (Lyon, France: International Agency for Research on Cancer) epidemiological overview; Br. Med. Bull. 52 3–11 pp 21–28 Whiteside G, Cougnon N, Hunt S P and Munglani R 1998 Thornberry N A and Lazebnik Y 1998 Caspases enemies within; An improved method for detection of apoptosis in Science 281 1312–1316 tissue sections and cell culture, using the TUNEL technique Tsujimoto Y 1998 Role of Bcl-2 family proteins in apoptosis: combined with Hoechst stain; Brain Res. Brain Res. Protoc. 2 apoptosomes or mitochondria? Genes Cells 3 697–707 160–164 Tuder R M, Petrache I, Elias J A, Voelkel N F and Henson P M Whiteside G and Munglani R 1998 TUNEL, Hoechst and 2003 Apoptosis and emphysema: the missing link; Am. J. immunohistochemistry triple-labeling: an improved method for Respir. Cell Mol. Biol. 28 551–554 detection of apoptosis in tissue sections—an update; Brain Res. US Department of Health and Human Services 1988 The health Brain Res. Protoc 3 52–53 consequences of smoking: nicotine addiction: a report of the Wright J L and Churg A 1995 Smoke-induced emphysema in Surgeon General (Rockville, MD: Center for Health Promotion guinea pigs is associated with morphometric evidence of and Education, Offi ce on Smoking and Health) DHHS collagen breakdown and repair; Am. J. Physiol. Lung Cell Mol. Publication No (CDC) 88-8406 Physiol. 268 L17–L20 US Department of Health and Human Services 1998 Reducing the Wright J L and Churg A 2002 A model of tobacco smoke- health consequences of smoking: 25 years of progress. A report induced airfl ow obstruction in the guinea pig; Chest 121 of the Surgeon General (Rockville, MD: US Department of 188S–191S Health, Centers for Disease Control, Center for Chronic Disease Wright J L, Farmer S G and Churg A 2002 Synthetic serine elastase Prevention and Health Promotion, Offi ce on Smoking and inhibitor reduces cigarette smoke–induced emphysema in Health) DHHS Publication No (CDC) 89-8411 guinea pigs; Am. J. Respir. Crit. Care Med. 166 954–960

MS received 9 December 2009; accepted 1 May 2010 ePublication: 12 May 2010

Corresponding editor: INDRANEEL MITTRA

J. Biosci. 35(2), June 2010