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Activated Charcoal Filter Effectively Reduces P-Benzosemiquinone From Activated charcoal fi lter prevents emphysema 217 Activated charcoal fi lter effectively reduces p-benzosemiquinone from the mainstream cigarette smoke 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, India *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 cigarettes 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 lung 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 smoking is the world’s single most preventable (PAHs) and tobacco-specifi c nitrosamines (TSNA). Most cause of disease and death. 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 lungs 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, nicotine is the primary source of tobacco and Helfant 1988; Sherman 1991; Wald and Hackshaw dependence (US Department of Health and Human 1996; US Department of Health and Human Services Services 1988). Others are toxins and carcinogens, 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 liquid 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 Hungary. 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 carbon monoxide (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 blend and consequent emissions. 2008). Wills Navy Cut 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
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