Activated charcoal ﬁ lter effectively reduces p ...

Activated charcoal fi lter prevents emphysema 217

J. Biosci. 35(2), June 2010

1. Introduction

Cigarette smoking is the world’s single most preventable

cause of disease and death. About one third of all adults in

the world are smokers (Slama 2008). Each year, over fi ve

million people throughout the world die from smoking-

related illness (IARC 2002). Cigarette smoke (CS) contains

more than 4000 compounds (Stewart and Kleihues 2003).

Among these, nicotine is the primary source of tobacco

dependence (US Department of Health and Human

Services 1988). Others are toxins and carcinogens, such

as free radicals, harmful gases, volatile organic compounds

(VOCs), aldehydes, polycyclic aromatic hydrocarbons

(PAHs) and tobacco-specifi c nitrosamines (TSNA). Most

of these chemicals are considered to be causative agents

for CS-induced life-threatening diseases, particularly

cancer of the lungs and other organs, myeloid leukaemia,

cardiovascular diseases and chronic obstructive pulmonary

disease (COPD), including bronchitis and emphysema (Shah

and Helfant 1988; Sherman 1991; Wald and Hackshaw

1996; US Department of Health and Human Services

1998; IARC 2002; World Cancer Report 2003; Harris et al.

http://www.ias.ac.in/jbiosci J. Biosci. 35(2), June 2010, 217–230, © Indian Academy of Sciences 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

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

Neekkan Dey et al.218


2004). Undoubtedly, the best way of preventing CS-induced

diseases is cessation of smoking. However, approaches to

cessation of smoking by public health campaigns and anti-

smoking laws passed by local governments have had limited

success. So, a practicable approach is to selectively reduce

the toxins from mainstream smoke. One such approach

is the use of cigarette fi lters. Among the fi lters, cellulose

acetate (CA) dominates the global fi lter market and charcoal

fi lters comprise about 10%. While a CA fi lter is effective

in reducing only a little portion of the tar, it is not at all

selective for the other toxins of cigarette smoke.

Charcoal-fi ltered (CF) cigarettes are predominantly

used (≈90%) in several countries, including Japan, Korea,

Venezuela and Hungary. It is less common (≈1%) in the

United States (Laugesen and Fowles 2006). However,

these charcoal fi lter designs have little effect on the

delivery of tar, nicotine and carbon monoxide (Polzin et al.

2008). Available data do not support the belief that use of

commercial charcoal-fi ltered cigarettes reduces the risk of

smoking-related diseases (Marugame et al. 2004; Muscat et

al. 2005; Laugesen and Fowles 2006). In particular, there do

not appear be any studies on the effect of CF cigarettes on

the prevention of COPD (Polzin et al. 2008; Marugame et

al. 2004; Muscat et al. 2005; Coggins and Gaworski 2008;

Han-Jae et al. 2009). It is known that cigarette smoking is

by far the commonest cause of COPD in western countries,

accounting for about 95% of cases (Lopez and Murray 1998;

Pauwels and Rabe 2004; Barnes et al. 2003). Emphysema

is a prominent pathological feature of COPD. Emphysema

is irreversible and currently there is no effective treatment

aimed at curing this fatal disease. This is particularly

because CS is a highly complex mixture of several thousand

compounds and it is not yet known whether a particular

compound or a number of compounds are responsible for

causing CS-induced emphysema. Once identifi ed, removal

of that chemical entity could effectively reduce smokers’

risk. Earlier, it was indicated that long-lived semiquinone

radical(s) present in an aqueous extract of CS (AECS) is

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

fragmentation are hallmarks of emphysema (Tuder et al.

2003). We have isolated a major semiquinone from AECS

and characterized it as p-benzosemiquinone by various

physicochemical analyses, including ultraviolet (UV),

mass, nuclear magnetic resonance (NMR) and electron

spin resonance (ESR) spectroscopy (Banerjee et al. 2008;

Chatterjee, US patent 2005; Chatterjee, Japan patent 2008;

Chatterjee, Korea patent 2008; Chatterjee, Europe patent

2008).

p-Benzosemiquinone (p-BSQ) is present in the

mainstream smoke of all cigarettes, irrespective of the

brands examined. Using various in vitro and in vivo analyses,

we had demonstrated that p-BSQ largely mimics AECS in

causing protein modifi cation, apoptosis and emphysematous

lung damage (Banerjee et al. 2008).

A conventional CA fi lter is ineffective in adsorbing p-

BSQ. We have also observed that two brands of commercial

charcoal-fi ltered cigarettes, Mild Seven Light and Magna,

containing uneven grain sizes of charcoal dispersed in CA

tow, are ineffi cient in reducing p-BSQ from mainstream

CS. In fact, elimination of p-BSQ from the smoke depends

on the amount and the particular grain size of the activated

charcoal used (Chatterjee, US Patent 2006). Using human

lung epithelial cells (A549) as well as a guinea pig model,

here we show that an activated charcoal fi lter designed by us

(Chatterjee, US patent 2005; Chatterjee, Japan patent 2008;

Chatterjee, Korea patent 2008) effectively reduces p-BSQ

from mainstream CS and thereby prevents cytotoxicity,

protein modifi cation, apoptosis and emphysematous lung

damage in guinea pigs.

2. Materials and methods

2.1 Chemicals and reagents

Activated charcoal (20–60 mesh) was purchased from

Sigma, USA. Granules of 60 mesh were separated by

grinding and sieving. An oxyblot protein oxidation detection

kit was purchased from Intergen Company, USA. The in

situ cell death detection kit was obtained from Roche, USA.

The kit for protein estimation was obtained from Bio-Rad,

USA. Antibodies against caspase 3, cleaved caspase 3, Bax,

Bcl-2, p53, phospho-p53, anti-rabbit horseradish peroxidase

(HRP)-conjugate and anti-mouse HRP-conjugate as well

as the chemiluminescent kit for immunoblot analysis

were obtained from Cell Signaling Technology, USA. The

Annexin V-FITC kit was purchased from BD Biosciences.

Anti-tubulin antibody was obtained from Santa Cruz

Biotechnology, Inc, USA. All other chemicals were of

analytical grade.

2.2 Cigarettes

Different brands of cigarettes were purchased from the

local market and used without delay. All the cigarettes used,

except Mild Seven Light (Japanese) and Magna (Russian),

were conventional CA fi lter-tipped. Mild Seven Light and

Magna were charcoal fi lter cigarettes. Our fi nding may have

limitations, because manufacturers might have changed a

brand’s design or tobacco blend and consequent emissions.

Wills Navy Cut is an Indian commercial cigarette. CA fi lter-

tipped Kentucky reference cigarettes were obtained from

the University of Kentucky Tobacco and Health Research

Institute (Lexington, Kentucky).



2.3 Preparation of charcoal-fi ltered cigarettes

Activated charcoal fi lter cigarettes used in the present

study were prepared from the original branded cigarettes

by replacing the conventional CA fi lter with our activated

charcoal fi lter using 150 mg charcoal granules of 60 mesh

(250 microns) (Chatterjee, US Patent 2006). Essentially,

the charcoal fi lter we developed is a cavity fi lter in which

the activated charcoal granules are placed in a void space

between two segments of CA fi lters. One portion of the CA

fi lter (≈14 mm) is the mouthpiece and other portion (≈3

mm) constitutes a barrier between the charcoal bed and the

tobacco portion. The CA mouthpiece was used to prevent

any leakage of charcoal granules in the mainstream smoke.

The various parts, i.e. the CA mouth piece, charcoal bed, thin

CA fi lter placed in between the charcoal bed and the tobacco

portion as well as the tobacco portion, were all constructed

in one single unit without any ventilation.

2.4 Preparation of aqueous extract of cigarette smoke

(AECS) solution

Smoke from one cigarette was extracted with 1 ml of 50 mM

potassium phosphate buffer, pH 7.4, fi ltered through a 0.22

μm Millipore fi lter and the pH adjusted to 7.4, as described

before in detail (Panda et al. 1999). The AECS thus obtained

was used immediately.

2.5 Isolation and characterization of p-benzosemiquinone

(p-BSQ)

p-BSQ was isolated from AECS by differential solvent

extraction, thin-layer chromatography (TLC) and high

performance liquid chromatography (HPLC) as described

earlier (Banerjee et al. 2008). p-BSQ was characterized

by various physicochemical analyses, including UV, mass,

NMR and ESR spectroscopy as reported earlier (Banerjee

et al. 2008).

2.6 Measurement of the comparative yields of p-BSQ in

smoke solution prepared from different cigarettes

p-BSQ in smoke solution was quantitatively measured by

HPLC as described earlier (Banerjee et al. 2008). About

5–10 μl of the smoke solution, fi ltered through a 0.22 μm

Millipore fi lter, was diluted about 40 times with mobile

solvent and 20 μl of this diluted solution was injected into

the HPLC (Simadzu 10A) with a UV detector set at 294

nm using a normal phase silica column (Lichrospher ®

Si60, Merck). The mobile solvent was methylene chloride:

methanol (90:10, v/v) with a fl ow rate of 0.5 ml/min.

The retention time of p-BSQ was 8.808 min. The amount of

p-BSQ present in the smoke solution was calculated from a

standard curve obtained with pure p-BSQ.

2.7 Measurement of protein damage

Protein damage was measured by carbonyl formation in

bovine serum albumin (BSA) after reaction with 2, 4-

dinitrophenyl hydrazine, similar to that done earlier in

our laboratory (Panda et al. 1999). The incubation system

contained 1 mg BSA and 50 μl of smoke solution obtained

from cigarettes with or without a charcoal fi lter in a fi nal

volume of 200 μl of 50 mM potassium phosphate buffer,

pH 7.4. After incubation for 1 h at 37°C, the protein was

precipitated with 200 μl of trichloroacetic acid solution and

the rest of the procedure followed was as described earlier

(Panda et al. 1999). The values are expressed as nmoles of

carbonyl formed per mg BSA.

2.8 Measurement of nicotine

Smoke from a lit cigarette was allowed to dissolve in 2 ml

of 50 mM potassium phosphate buffer, pH 7.4 and fi ltered

through a 0.22 μm Millipore fi lter. One millilitre of the

yellow coloured fi ltrate was extracted with 1 ml of methylene

chloride by vigorous vortexing to extract the nicotine in the

methylene chloride layer. Of the methylene chloride layer

containing the nicotine, 500 μl was then vortexed with 500 μl

of 50 mM HCl solution and the nicotine in the aqueous layer

was estimated by HPLC analysis at 254 nm (Chatterjee, US

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

solution was injected into the HPLC column. A standard

solution of nicotine was prepared in a similar manner and

analysed. The parameters used were: Instrument, Shimadzu

10A; Column, Lichrospher® 100 RP-18 endcapped (5

μm), Merck; mobile solvent: 50 mM KH2PO4 solution:

accetonitrile:methanol (78:17: 5, v/v) containing 1 mM

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

analysis under these conditions was 10 ng.

2.9 Measurement of tar

Tar was collected from the mainstream smoke by suction (30

cm water) through a Millipore fi lter unit. The Millipore fi lter

(0.22 μm) was changed every 2 min to avoid clogging of the

fi lter. For each cigarette, 4 fi lters were used. After complete

burning of the tobacco, the fi lters were dried in a vacuum

desiccator and weighed. The difference in weight of the

fi lters before and after collecting the particulate portion was

the weight of the tar (Chatterjee, US patent 2005).

2.10 Cell culture

A549 cells were grown to 50–60% confl uence in HamF12

medium containing 10% foetal calf serum (GIBCO-BRL,

USA), 100 units /ml penicillin, 100 μg/ml streptomycin

and 4 mM glutamine/ml. The cells were grown at 37°C in a

humifi ed incubator maintained in an atmosphere of 95% air

and 5% CO2.

2.11 Cytotoxicity assay

The cytotoxicity of the aqueous extract of mainstream smoke

from CA-fi ltered and charcoal-fi ltered (CF) cigarettes was

evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) assay (Mosmann 1983). After

respective treatments with 50 μl/ml of the CA or CF AECS

solution for 1 h, the culture medium was replaced by medium

containing 0.5 mg/ml MTT and incubated for an additional

3 h. The blue MTT formazan was dissolved in 1 ml of

dimethyl sulphoxide (DMSO), and the absorbance values

were determined at 560 nm in a UV-VIS spectrophotometer

(Shimadzu UV-2540).

2.12 Differentiation between apoptosis and necrosis by

FACS

In the early stages of apoptosis, changes occur in the

plasma membrane. One of the cell surface alterations is

the translocation of phosphatidyl serine (PS) from the inner

surface of the cell membrane to the outer layer. Annexin V,

a Ca2+-dependent phospholipid-binding protein with high

affi nity for phosphatidyl serine, is a sensitive probe for PS

and can thereby detect early apoptotic cells in a live cell

population. Propidium iodide (PI) stains only the necrotic

cells. To distinguish between apoptosis and necrosis, A549

cells (3 x 106) treated with 50 μl of CA-fi ltered or CF AECS

solution for 12 h were stained by PI and Annexin V-FITC

(Becton Dickinson) according to manufacturer’s protocols

and analysed using the FACS Calibur-Cell Quest software

(Becton Dickinson). A total of 10 000 events were acquired.

The cells were properly gated and a dual parameter dot plot

of FL2H (X-axis; PI fl uorescence, linear scale) vs FL1-H (Y-

axis; FITC-fl uorescence, logarithmic scale) was recorded.

2.13 Terminal deoxynucleotidyl transferase-mediated de-

oxyuridine triphosphate nick end labelling (TUNEL) assay

A549 cells (2 x 106) treated with 50 μl of AECS were fi xed

with 4% p-formaldehyde and permeabilized with titron X-

100 (0.1%) in 0.1% Na-citrate. The cells were then washed

with phosphate buffered saline and subjected to TUNEL

assay using an in situ cell death detection kit (Roche, USA)

according to the manufacturer’s instruction. The stained cells

were counted under a fl uorescence microscope (Olympus

B). Nuclei were simultaneously counted by counterstaining

with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)

(Santa Cruz Biotechnology, USA). The percentage of

TUNEL-positive cells was calculated (Banerjee et al. 2008).

2.14 Exposure of guinea pigs to cigarette smoke (CS)

The procedure we followed was essentially similar to that

described earlier in detail (Banerjee et al. 2007, 2008). Male

short-hair guinea pigs weighing 400–500 g were used for

all the experiments. All animal treatment procedures met

the Institutional Animal Ethics Committee guidelines. The

guinea pigs were fed a vitamin C-free diet for 7 days to

minimize the vitamin C level of tissues (Panda et al. 2000).

This is because vitamin C is a potential inhibitor of CS-

induced protein oxidation (Panda et al. 1999, 2000), which

would otherwise counteract the damaging effect of CS. The

vitamin C-free diet given to the guinea pigs was similar to that

described earlier (Misra et al. 2003). After 7 days of vitamin

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

(Banerjee et al. 2007), along with oral supplementation of 1

mg vitamin C/animal/day for 2 weeks (6 days a week, Sunday

excluded). Deprivation of vitamin C was discontinued to

avoid the onset of scurvy. One mg of vitamin C per day is

approximately the minimum dose needed to prevent scurvy

in the guinea pig (Banerjee et al. 2007). With this dose of

vitamin C, there was no symptom of onset of scurvy in any of

the guinea pigs during the experimental period. The smoke

chamber was similar to that of a vacuum desiccator with an

open tube at the top and a side tube fi tted with a stopcock, as

described earlier (Banerjee et al. 2007, 2008). The volume

of the chamber was 2.5 l. The cigarette placed at the top was

lit and CS was introduced into the chamber containing the

guinea pig by applying a mild suction of 4 cm water through

the side tube for 5 s. Thereafter, the vacuum was turned off

and the guinea pig was further exposed to the accumulated

smoke for another 40 s. The total duration of exposure to

smoke from one puff was thus 45 s. Altogether, 2 puffs per

cigarette were given, allowing the animal 1 min rest in a

smoke-free atmosphere to breathe air between each puff.

The gap between one cigarette and the next was 1 h. Pair-fed

sham controls were subjected to air exposure instead of CS

under similar conditions.

The guinea pigs were divided into the following

experimental groups (N = 6/group): (i) exposed to air, (ii)

exposed to CA-fi ltered CS, (iii) exposed to charcoal-fi ltered

CS. After exposure to air or CS for up to 15 days, both the

sham controls and the CS-exposed guinea pigs were deprived

of food overnight and sacrifi ced next day by diethyl ether



inhalation. The lungs were then excised immediately and

processed for analysis.

2.15 Histology of lung section for measurement of

emphysematous lung damage

Lung damage caused by exposure to CS was quantifi ed by

measuring the mean linear intercept (Lm) and destructive

index (DI). Lm represents the average size of alveoli,

indicating the air space, which is increased in emphysema.

It was measured by the technique originally described

by Dunnill (1962). Images randomly selected from

haematoxylin–eosin (H&E) stained lung sections were

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.

Briefl y, a cross-hair grid consisting of horizontal and vertical

lines (1 cm apart) was laid over the digital image on the

computer screen. The number of times each line crossed an

alveolar wall, both horizontally and vertically, was manually

counted. Results are expressed by the formula:

Lm = L/(X×m),

where Lm = mean linear intercept; L= total length of the

lines in mm; X = magnifi cation factor and m = sum of

intercepts (the points where the horizontal and vertical

lines independently intercepted the alveolar walls). Four

independent lung sections per animal were analysed.

The degree of destruction of the alveolar walls was

quantifi ed by measuring the DI following the microscopic

manual point count method of Saetta et al. (1985) using the

Dewinter Biowizard 4.1 software, as stated above, except

that the lines of the cross-hair grid were 2 cm apart. The

spaces directly under the cross-hair points were counted as

either normal (N) or destroyed (D). The DI was computed

from the formula:

DI = D/(D+N) ×100(%),

where D indicates destroyed and N indicates normal points.

An alveolar space was considered to be normal if it was

surrounded by intact walls or by wall disrupted in only one

place. Alveolar space was considered to be destroyed when

the wall of an alveolus was disrupted in two or more places.

2.16 Western blot

For western blot, cells (3 x 106) were treated with CA-

fi ltered or charcoal-fi ltered AECS solution for 1 h and then

reincubated in fresh Ham F12 medium for 16 h. Control cells

received no treatment. After that, cell extracts were prepared

by lysing the cells in lysis buffer (20 mM Tris [tris(hydroxy

methyl)aminomethane chloride], pH 7.4; 250 mM NaCl; 2

mM EDTA [ethylenediaminetetraacetic acid], pH 8.0; 0.1%

Triton-X100; 0.01 mg/ml aprotinin; 0.005 mg/ml leupeptin;

0.4 mM phenylmethanesulphonyl fl uoride [PMSF]; and 4

mM NaVO4). Lysates were then centrifuged at 20 000 g for

10 min to remove insoluble material. The supernatant (30–50

μg protein) was resolved on 12% sodium dodecyl sulphate-

polyacrylamide gel electrophoresis (SDS-PAGE) gel. After

electrophoresis, the proteins were electrotransferred to a

PVDF membrane, blocked with 5% non-fat milk (Bio-

Rad), and probed with antibodies against caspase 3, cleaved

caspase 3, Bax, Bcl-2, p53 and phospho-p53 (1:1000) for 1 h.

Thereafter, the blot was washed, exposed to HRP-conjugated

secondary antibodies for 1 h, and fi nally detected by

chemiluminescence, as done earlier (Banerjee et al. 2008).

2.17 Statistical analysis

All values are expressed as mean ± SD. Statistical

signifi cance was carried out using one-way ANOVA. The P

values were calculated using appropriate F-tests. Difference

with P values <0.05 were considered signifi cant.

3. Results

3.1 Charcoal fi lter effectively reduces p-BSQ from main-

stream smoke of different brands of cigarettes and prevents

protein carbonyl formation

Table 1 shows that mainstream smoke from different brands

of CA-fi ltered commercial cigarettes as well as Kentucky

research cigarettes contain high amounts of p-BSQ. When

the CA fi lters are replaced by charcoal fi lters, the p-BSQ

content is reduced by 73–80%. Earlier, we had reported that

p-BSQ from CS is a major toxic component that is largely

responsible for carbonyl formation in proteins (Banerjee

et al. 2008). Table 1 indicates that when the CA fi lter is

replaced by a charcoal fi lter, carbonyl formation in BSA

is markedly reduced. Table 1 further shows that tar and

nicotine delivery are also considerably reduced in smoke

from charcoal-fi ltered cigarettes. In a separate experiment,

we have observed that fortifi cation of tobacco (1 g) with 3

mg nicotine per cigarette leads to increased nicotine delivery

in the mainstream smoke to almost the original level (data

not shown). However, such fortifi cation does not cause any

increase in the p-BSQ content of the smoke, apparently

because nicotine is not a precursor of p-BSQ.

3.2 Charcoal fi lter prevents AECS-induced alteration of

morphology and loss of viability in A549 cells

In the pathogenesis of CS-induced pulmonary diseases,

injury of the alveolar epithelium is an important process.





We compared the effects of CA-fi ltered AECS with

charcoal-fi ltered AECS on the morphology and viability of

A549 cells. Examination under a phase-contrast microscope

showed that exposure of the cells to CA-fi ltered AECS

caused an increase in fl oating cells accompanied by a

decrease in the density of cells. The cells still attached

became round in shape and the gap between the cells was

enlarged (fi gure 1A, middle panel). However, when the

cells were exposed to charcoal-fi ltered AECS, both the

confl uence and the morphology were almost similar to that

of control cells (fi gure 1A, fi rst and third panels). Similar

observations were made regarding the cytotoxicity of AECS.

MTT assay revealed that compared with the normal controls,

CA-fi ltered AECS reduced the viability of the cells to about

32%. In contrast to this, the viability of charcoal-fi ltered

AECS-treated cells was about 78% (fi gure 1B).

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

or without

charcoal fi lter

Brand of

cigarette

p-BSQ†

content (μg)

Per cent

reduction in

p-BSQ

content**

BSA* oxidation

(nmoles of

carbonyl

formed)#

Nicotine‡

delivery (mg)

Tar¶ content

(mg)

1 Without

With

Kentucky

3R4F†

100 ± 7.07

20 ± 1.79

--

80

6.0

1.5

0.75

0.40

9

4

2 Without

With

Kentucky

1R3F†

180 ± 17.90

40 ± 2.83

--

78

10.0

3.0

1.16

0.50

15

10

3 Without

With

Wills

Navy Cut§

200 ± 17.90

40 ± 3.16

--

80

10.0

2.5

1.00

0.45

20

12

4 Without

With

Winston 200 ± 20.00

45 ± 4.47

--

77

12.4

3.0

1.3

0.55

18

11

5 Without

With

Camel 180 ± 16.10

45 ± 8.94

--

75

10.6

2.8

1.3

0.52

17

10

6 Without

With

Viceroy 175 ± 13.40

40 ± 4.56

--

73

10.4

2.7

1.3

0.54

16

9

7 Without

With

Marlboro 170 ± 13.40

36 ± 3.44

--

79

10.0

2.5

1.2

0.50

16

10

8 Without

With

Benson &

Hedges

170 ± 14.30

34 ± 3.58

--

80

10.2

2.5

1.1

0.47

16

9

9 Without

With

Virginia

Slims

160 ± 14.30

34 ± 4.47

--

79

9.8

2.4

1.1

0.46

14

8

10 Without

With

Cambridge 160 ± 13.40

36 ± 4.50

--

77

10.0

2.6

1.0

0.44

15

9

11 Without

With

Kent 150 ± 9.80

30 ± 2.83

--

80

8.5

2.2

1.0

0.46

14

9

12 Without

With

Kool 155 ± 11.40

32 ± 4.56

--

79

9.0

2.0

1.0

0.44

14

9

13 Without

With

Classic 135 ± 8.94

25 ± 3.69

--

79

7.5

1.6

0.8

0.40

12

8

14 Without

With

Monte Carlo 140 ± 9.88

25 ± 2.68

--

78

7.0

1.5

0.8

0.40

12

8

15 Without

With

Mild

Seven Light*

100 ± 7.07

20 ± 2.28

--

80

6.0

1.4

0.7

0.38

8

3

16 Without

With

Magna* 110 ± 8.10

22 ± 2.83

--

80

6.5

1.5

0.6

0.36

9

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.



3.3 Charcoal fi lter prevents AECS solution-induced apop-

tosis in A549 cells as evidenced by FACS analysis using

Annexin V and PI

Flowcytometric data (fi gure 2) indicated that when A549

cells were treated with 50 μl/ml medium of CA-fi ltered

AECS solution there was a signifi cant increase (78.92%)

of Annexin V-positive cells compared with the untreated

control cells (1.49%; P<0.05). This indicates that the CA-

fi ltered AECS-treated cells were entering into the apoptotic

phase. However, when the cells were treated with 50 μl/ml

medium charcoal-fi ltered AECS solution, the percentage of

Annexin V-positive cells was only 2.18%, indicating that the

charcoal fi lter prevented AECS solution-induced apoptosis

in A549 cells. In all the cases, the percentage of PI-positive

necrotic cells were negligible.

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.



3.4 Charcoal fi lter prevents AECS-induced DNA fragmen-

tation of A549 cells as evidenced by TUNEL assay

When A549 cells were exposed to CA-fi ltered AECS (50

μl/ml medium), the percentage of TUNEL-positive nuclei

markedly increased (90 ± 5 SD), as indicated by green

fl uorescence attributable to fl uorescein-dUTP labelling

(fi gure 3 A, B, upper panel). The lower panel shows the

nuclei counterstained with DAPI. In contrast, there was

little increase (4 ± 1 SD) in TUNEL-positive cells exposed

to charcoal-fi ltered AECS (fi gure 3 A, B). This indicates

that AECS-induced oligonucleosomal fragmentation is

prevented by the charcoal fi lter.

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

CA-fi ltered AECS on A549 cells is caused by an increase of

activated p53 as well as an increased Bax/Bcl-2 ratio, which

is completely prevented by the charcoal fi lter.

CA-fi ltered AECS-induced apoptosis was further

supported by checking the level of cleaved caspase 3 by

western blotting of A549 cell lysate using anti-caspase

3 antibody (fi gure 4B, panel I). The level of the cleaved

product of caspase 3 (17 kDa) was markedly increased

(fi gure 4B, panel II, lanes 4 and 5) in response to CA-fi ltered

AECS treatment (30 μl/ml or 50 μl/ml medium). There

was no activation of caspase 3 in the case of the untreated

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,

respectively; lanes 2 and 3).

3.6 Smoke from charcoal-fi ltered cigarettes prevents

emphysema as evidenced by air space enlargement and

parenchymal destruction

Histology profi les showed that when the guinea pigs are

exposed to CA-fi ltered CS for two weeks at an exposure rate

of 5 cigarettes (2 puffs/cigarette)/guinea pig/day, there was

marked emphysematous damage of the lung, as compared

with sham control guinea pigs exposed to air (fi gure 5 A,

B). The damage was evidenced by morphometric change

Figure 2. Charcoal fi lter prevents aqueous extract of cigarette

smoke (AECS) solution-induced apoptosis in A549 cells as

evidenced by FACS analysis using Annexin V and propidium

iodide (PI) double staining technique. After treatment with

cellulose acetate (CA)-fi ltered AECS solution and charcoal-fi ltered

AECS solution, A549 cells were labelled with PI and AnnexinV-

FITC and then analysed on a fl owcytometer. Controls were cells

without treatment with AECS, but double-stained with PI and

AnnexinV-FITC. Details are given in the Materials and methods

section 2.12. Dual parameter dot plot of FITC fl uorescence (Y-axis)

vs PI (X-axis) has been shown as fl uorescence intensity. Quadrants:

lower left, viable cells; upper left, apoptotic cells; upper right, late

apoptotic and lower right, necrotic cells.



and enlargement of air spaces. In contrast, when the guinea

pigs were exposed to smoke from charcoal-fi ltered (CF)

cigarettes, no apparent lesion in the lung cells was observed

and the histological profi le appeared to be similar to that of

normal guinea pigs (fi gure 5 C). Quantitative evaluation

of lung damage was done by measuring the mean Lm and

DI of the infl ated lung sections (table 2). The number of

guinea pigs used in each group was eight. Four representative

non-overlapping images from each lung section

were captured in an Olympus B microscope attached

with a CCD Cool camera and analysed by the Dewinter

Biowizard 4.1 software. Sixteen images were analysed

in each group. Table 2 shows that compared with the

Lm (0.379 ±0.009) and DI (35.3 ± 7.2) of normal guinea

pigs, both the Lm and DI (0.449 ± 0.045 and 58.2 ± 5.4,

respectively) of CA-fi ltered CS-exposed guinea pigs are

signifi cantly increased (P<0.05). In contrast, there is no

signifi cant difference between the Lm (0.390 ± 0.020) and

DI (38.8 ± 6.3) of the lung sections of guinea pigs exposed

to charcoal-fi ltered CS and those of sham controls exposed

to air (P>0.05). The results confi rm that CS-induced

emphysema is signifi cantly prevented when the CA fi lter is

replaced by a charcoal fi lter.

4. Discussion

Emphysema is a prominent feature of COPD, which is

a major and increasing global cause of mortality and

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.



morbidity (Lopez and Murray 1998; Pauwels and Rabe

2004). Cigarette smoking is by far the commonest cause of

emphysema in western countries, accounting for about 95%

of cases (Barnes et al. 2003). The cellular and molecular

mechanisms of CS-induced emphysema remain unclear,

particularly because CS contains about 4000 compounds

(Stewart and Kleihues 2003). Earlier, we had isolated p-BSQ,

a long-lived radical from an Indian commercial CA-fi ltered

CS, and demonstrated that p-BSQ largely mimics CS-

induced protein modifi cation, apoptosis and emphysematous

lung damage in a guinea pig model (Banerjee et al. 2008). In

this study, we have shown that besides the Indian cigarette,

substantial amounts of p-BSQ are present in the mainstream

smoke of all the CA-fi ltered cigarettes studied, irrespective

of the brand. We also examined Kentucky reference

research cigarettes, whose tobacco blend, nicotine content

and toxicity have been reported to be representative of other

cigarettes (Han-Jae et al. 2009; Doolittle et al. 1990; Chen

and Moldoveanu 2003).

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

by fortifi cation of the tobacco with nicotine. Fortifi cation by

nicotine does not cause any increase in the p-BSQ content,

apparently because nicotine is not a precursor of p-BSQ.

Nicotine is a pharmacological component and a primary

source of tobacco dependence. The amount of nicotine

present in the CS of a cigarette does not appear to be a cause

of CS-induced toxicity and apoptosis (Ramage et al. 2006).

It has been reported that the semiquinone of CS is

cytotoxic (Chouchane et al. 2006; Pryor et al. 1998). Here

we show that a charcoal fi lter, which effectively reduces the

p-BSQ of mainstream smoke, also causes a marked reduction

in the cytotoxicity of CS. Cytotoxicity was determined by

microscopic examination of cell morphology and viability

by MTT assay.

Previous observations from our laboratory had indicated

that the initial event of exposure of lung cells to CA-fi ltered

CS is protein damage, which is followed by apoptosis

(Banerjee et al. 2008). Once protein damage is prevented,

apoptosis is also prevented (Banerjee et al. 2008). Earlier,

we had demonstrated that p-BSQ largely mimics AECS-

induced apoptosis. Here we show that reduction of p-BSQ

from mainstream smoke by a charcoal fi lter is accompanied

by prevention of apoptosis. Apoptosis is an important mode

of cell death under both physiological and pathophysiological

conditions. Several techniques are available for the study

and quantitation of apoptosis in cell culture. Two commonly

used techniques to quantify apoptosis are: (i) FACS

analysis using Annexin V and (ii) measurement of DNA

fragmentation using the TUNEL method (Rao et al. 1998;

Whiteside et al. 1998; Whiteside and Munglani 1998). Here,

we show using both the techniques that apoptosis produced

Figure 4. (A) Immunoblot of phosphorylated p53, p53, Bax and

Bcl-2 in cell lysate of A549 cells treated with charcoal-fi ltered

and cellulose-acetate (CA)-fi ltered aqueous extract of cigarette

smoke (AECS) solution. Lane 1, untreated control; lanes 2 and

3, cells treated with 30 μl and 50 μl charcoal-fi ltered AECS/ml,

respectively; lanes 4 and 5, cells treated with 30 μl and 50 μl

CA-fi ltered AECS/ml, respectively. (B) Immunoblot of caspase 3

and cleaved caspase 3 in cell lysate of A549 cells treated with CF

and CA-fi ltered AECS solution. Lane 1, untreated control; lanes

2 and 3, treated with 30 μl and 50 μl CF-AECS/ml, respectively;

lanes 4 and 5, treated with 30 μl and 50 μl CA-fi ltered AECS/ml,

respectively. α-tubulin was used as the loading control.



in A549 cells by AECS prepared from CA-fi ltered cigarette

smoke is prevented when the CA fi lter is replaced by a

charcoal fi lter.

Caspases are aspartate-directed cysteine proteases with a

pivotal role in apoptosis. Caspase 3 is an important caspase

in the execution of downstream events in apoptosis. Caspases

contribute to apoptosis through disassembly of cell structures

by disrupting the nuclear structure and cleaving several

cytoskeletal proteins (Schraufstatter et al. 1984; Thornberry

and Lazebnik 1998). Caspases, synthesized initially as

inactive single polypeptide chains, undergo proteolytic

cleavage to produce subunits (cleaved caspase 3, e.g. 17 kDa)

having protease activity. We have shown that while cleaved

caspase 3 is produced by using a CA fi lter, it is not produced

when the CA fi lter is replaced by a charcoal fi lter.

Apoptosis is regulated by the expression of Bax, a

member of the Bcl-2 protein family (Kluck et al. 1997;

Tsujimoto 1998). Bax is pro-apoptotic and Bcl-2 is anti-

apoptotic. Here, we show that exposure of A549 cells to CA-

fi ltered AECS results in overexpression of Bax, but no such

overexpression takes place when the CA fi lter is replaced by

a charcoal fi lter. The level of Bcl-2 protein remains unaltered

in both the cases.

It is known that the phosphorylated form of p53

increases in response to DNA damage (Banin et al. 1998).

Using TUNEL assay, we have shown that marked DNA

fragmentation occurs by exposure of A549 cells to CA-

fi ltered AECS. We have further shown that such exposure

also causes an increase in phospho-p53. No such increase

in phospho-p53 occurred when the cells were exposed to

charcoal-fi ltered AECS.

Previously, we had shown that exposure of guinea

pigs to smoke from an Indian CA-fi ltered cigarette

causes emphysematous lung damage (Banerjee et al.

2007, 2008). Emphysema is defi ned as the ‘abnormal

permanent enlargement of the airspaces distal to the

terminal bronchioles, accompanied by destruction of their

walls’ (American Thoracic Society 1995). The validity

of a potential animal model of emphysema is tested by

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

destructive index (DI) of lung sections of normal and cigarette

smoke-exposed guinea pigs

Condition of exposure Mean linear

intercept (Lm)

Mean ± SD

Destructive index

(DI) Mean ± SD

Air (normal) 0.379 ± 0.009 35.3 ± 7.2

Smoke from cellulose

acetate-fi ltered

cigarettes§

0.449 ± 0.045 58.2 ± 5.4

Smoke from charcoal-

fi ltered cigarettes§

0.390 ± 0.020 38.8 ± 6.3

§Wills Navy Cut (Indian commercial cigarette) The number of

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

sections from the middle lobe of both the left and right lungs

were stained with haematoxylin and eosin. Four representative

non-overlapping images from each section were captured in an

Olympus B microscope attached with a CCD Cool camera and

analysed by the Dewinter Biowizard 4.1 software. Altogether

16 images (approximately 1600 point counts) were analysed in

each group. Details are given in Materials and methods. P values

between cellulose acetate-fi ltered smoke exposed and normal (air

exposed, normal) are: for Lm, P = 0.0039; DI, P = 0.0001 (highly

signifi cant) and that between charcoal-fi ltered smoke exposed

and normal are: for Lm, P = 0.27; DI = 0.80 (not signifi cant).



quantitative histopathological methods measuring both

airspace enlargement and destruction of the alveolar walls.

Measurements of Lm and DI are authentic parameters for

quantitation of enlargement of airspaces and destruction

of the alveolar walls (Robbesom et al. 2003; Saetta et al.

1985; Fehrenbach 2006). We have shown that both the Lm

and DI of lung sections produced by exposure of guinea

pigs to smoke from a CA-fi ltered cigarette are signifi cantly

increased. However, when the CA fi lter is replaced by a

charcoal fi lter, the values of Lm and DI are comparable with

those of normal sham control guinea pigs, indicating that

a charcoal fi lter prevents CS-induced emphysematous lung

damage in guinea pigs.

A number of reports from other laboratories indicate

that induction of emphysematous lesions in guinea

pigs by CS requires exposure times of at least several

months (Wright and Churg 1995, 2002; Wright et al. 2002).

In contrast to this, we produced emphysema within 14

days of CS exposure. The apparent difference is because

we used vitamin C-restricted guinea pigs to minimize

the vitamin C level in the tissues (Banerjee et al. 2007).

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.

2008).

In conclusion, we report that irrespective of the brand

of CA-fi ltered cigarettes examined, the mainstream smoke

contains p-BSQ, a major, highly toxic, long-lived water

soluble radical. The amount of p-BSQ varies with the

tar content. We have examined 14 brands of CA-fi ltered

cigarettes from different countries of the world, including

India, England, USA, as well as Kentucky Research

Cigarettes and 2 brands of CF-fi ltered cigarettes from

Japan and Russia and observed that smoke from all the

cigarettes contains substantial amounts of p-BSQ (100–200

μg/cigarette). This indicates that p-BSQ is a prominent toxic

component of CS, irrespective of the source of the cigarette.

Previous reports from our laboratory in human lung epithelial

cells (A549) and in vivo in guinea pigs indicate that p-BSQ

from CA-fi ltered CS causes protein damage, apoptosis and

emphysematous lung lesions (Banerjee et al. 2008). Here we

show that when the CA fi lter is replaced by a charcoal fi lter

developed in our laboratory, p-BSQ is markedly reduced

from the mainstream smoke and all these pathophysiological

events are prevented.

Apparently, the results obtained with guinea pigs may be

extrapolated to human smokers. The structure of the guinea

pig lung is similar to that of human lung (Wright and Churg

2002). In addition, the guinea pig develops morphological

and pathophysiological alterations after exposure to CS in the

same pattern as humans (Wright and Churg 2002). However,

the present study has some limitations. The results obtained

with guinea pigs should be validated by epidemiological

studies. Until now, limited epidemiological data are

available to demonstrate a conclusive benefi cial effect of

commercial CF cigarettes (Coggins and Gaworski 2008). In

our study, all the guinea pigs exposed to CA-fi ltered CS had

emphysematous lesions, whereas only about 15% of smokers

develop emphysema (Snider et al. 1985). So, some genetic

predisposition and nutritional status of vitamin C might be

involved in the susceptibility of a smoker to emphysema.

Nevertheless, since there is no curative therapy available

for emphysema, a practical approach would be prevention.

If the results obtained with guinea pigs are applicable to

humans, use of a charcoal fi lter developed in our laboratory

may protect smokers from emphysema.

Acknowledgements

The authors acknowledge fi nancial support from Council of

Scientifi c and Industrial Research, New Delhi, for carrying

out this research. ND is a Juthika Research Fellow; AD is

a Phulrenu Guha Research Fellow; AG is an ICMR Senior

Research Fellow and IBC is an INSA Honorary Scientist.

References

American Thoracic Society 1995 Standards for the diagnosis and

care of patients with chronic obstructive pulmonary disease;

Am. J. Respir. Crit. Care Med. 152 S77–S121

Banerjee S, Maity P, Mukherjee S, Sil A K, Panda K, Chattopadhyay

D J and Chatterjee I B 2007 Black tea prevents cigarette smoke-

induced apoptosis and lung damage; J. Infl ammation 4 3

Banerjee S, Chattopadhyay R, Ghosh A, Koley H, Panda K, Roy

S, Chattopadhay D J and Chatterjee I B 2008 Cellular and

molecular mechanisms of cigarette smoke-induced lung damage

and prevention by vitamin C; J. Infl ammation 5 21

Banin S, Moyal L, Shieh S, Taya Y, Anderson C W, Chessa

L, Smorodinsky N I, Prives C et al. 1998 Enhanced

phosphorylation of p53 by ATM in response to DNA damage;

Science 281 1674–1677

Barnes P J, Shapiro S D and Pauwels R A 2003 Chronic obstructive

pulmonary disease: molecular and cellular mechanisms; Eur.

Respir. J. 22 672–688

Chatterjee I B 2005 Process for the isolation of a major harmful

oxidant from cigarette smoke (US Patent No. 6,929,012)

Chaterjee I B 2008 Process for the isolation of a major harmful

oxidant from cigarette smoke (Korea Patent No. 10-0868687)

Chatterjee I B 2008 Process for the isolation of a major harmful

oxidant from cigarette smoke (Japan Patent No. 4094545)

Chatterjee I B 2006 Activated charcoal fi lter for effectively

reducing p-benzosemiquinone from the mainstream cigarette

smoke (US Patent No. 7,025,067 B2)Chatterjee I B 2008 Japan

Patent No. 3966856 Activated charcoal fi lter for effectively

reducing p-benzosemiquinone from the mainstream cigarette

smoke

Chatterjee I B 2008 Europe Patent No. EP1434503 (validated in UK,

Italy, France, Spain, Germany, Portugal and Greece) Activated



charcoal fi lter for effectively reducing p-benzosemiquinone

from the mainstream cigarette smoke

Chen P X and Moldoveanu S C 2003 Mainstream smoke chemical

analysis for 2R4F Kentucky reference cigarette; Beitr.

Tabakforsch. 20 448–458

Chouchane S, Wooten J B, Tewes F J, Wittig A, Müller B P, Veltel

D and Diekmann J 2006 Involvement of semiquinone radicals in

the in vitro cytotoxicity of cigarette mainstream smoke; Chem.

Res. Toxicol. 19 1602–1610

Coggins C R E and Gaworski C L 2008 Could charcoal fi ltration

of cigarette smoke reduce smoking-induced diseases? A review

of the literature; Regulatory Toxicology and Pharmacology 50

359–365

Doolittle D J, Lee C K, Ivett J L, Mirsalis J C, Ricco E, Rudd C

J, Burger G T and Hayes A W 1990 Comparative studies on

the genotoxic activity of mainstream smoke condensate from

cigarettes which burn or only heat tobacco; Environ. Mol.

Mutagen. 15 93–105

Dunnill M S 1962 Quantitative methods in the study of pulmonary

pathology; Thorax 17 320–328

Fehrenbach H 2006 Animal models of pulmonary emphysema: a

stereologist’s perspective; Eur. Resp. Rev. 15 136–147

Han-Jae S, Hyung-Ok S, Jung-Ho H, Chul-Hoon P, Hyeong-Seok

L, Dong-Wook L, Keon-Joong H and Hak-Chul H 2009 Effect

of cigarette fi lters on the chemical composition and in vitro

biological activity of cigarette mainstream smoke; Food Chem.

Toxicol. 7 192–197

Harris J E, Thun M J, Mondul M L and Calle E E 2004 Cigarette

tar yields in relation to mortality from lung cancer in the caner

prevention study II prospective cohort, 1982–8; BMJ 328

72–76

International Agency for Research on Cancer 2002 IARC

monographs on the evaluation of the carcinogenic risk of

chemicals to humans (Lyon, France: IARC) p. 83

Kluck R M, Bosy-Wetzel E, Green D R and Newmeyer D D 1997

The release of cytochrome c from mytochondria: a primary site

for Bcl-2 regulation of apoptosis; Science 276 1132–1136

Laugesen M and Fowles J 2006 Marlboro UltraSmooth: a

potentially reduced exposure cigarette?; Tob. Control 15

430–435

Lopez A D and Murray C C 1998 The global burden of disease,

1990–2020; Nat. Med. 4 1241–1243

Marugame T Sobue T, Nakayama T Suzuki T, Kuniyoshi H,

Sunagawa K, Genka K, Nishizawa N et al. 2004 Filter cigarette

smoking and lung cancer risk; a hospital-based case–control

study in Japan; Br. J. Cancer 90 646–651

Misra A, Chattopadhyay R, Banerjee S, Chattopadhyay D J and

Chatterjee I B 2003 Black tea prevents cigarette smoke-induced

oxidative damage of proteins in guinea pigs; J. Nutr.133

2622–2628

Mosmann T 1983 Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays; J.

Immunol. Methods 65 55–63

Muscat J E, Takezaki T, Tajima K and Stellman SD 2005 Charcoal

cigarette fi lters and lung cancer risk in Aichi Prefecture; Japan

Cancer Sci. 96 283–287

Panda K, Chattopadhyay R, Ghosh M K, Chattopadhyay D J and

Chatterjee I B 2001 Cigarette smoke-induced protein oxidation

and proteolysis is exclusively caused by its tar phase: prevention

by vitamin C; Toxicol. Lett. 123 21–23


Chatterjee I B 1999

Vitamin C prevents cigarette smoke induced oxidative damage of

proteins and increased proteolysis; Free Radic. Biol. Med. 27

1064–1079


Chatterjee I B 2000

Vitamin C prevents cigarette smoke induced oxidative damage in

vivo; Free Radic. Biol. Med. 29 115–124

Pauwels R A and Rabe K F 2004 Burden and clinical features of

chronic obstructive pulmonary disease (COPD); Lancet 364

613–620

Pryor W A, Deoley M M and Church D F 1986 The inactivation of

α1-proteinase inhibitor by gas-phase cigarette smoke: protection

by antioxidants and reducing species; Chem. Biol. Interact. 57

271–283

Pryor W A, Prier D G and Church D F 1983 Electron spin resonance

study of mainstream and side stream cigarette smoke: nature of

the free radicals in gas phase smoke and cigarette tar; Environ.

Health Perspect. 47 345–355

Pryor W A, Stone K, Zang L Y and Bermudez E 1998 Fractionation

of cigarette tar extracts: fractions that contain the tar radical

cause DNA damage; Chem. Res. Toxicol. 11 441–448

Polzin G M, Zhang L, Hearn B A, Tavakoli A D, Vaughan C, Ding

Y S, Ashley D L and Watson C H 2008 Effect of charcoal-

containing cigarette fi lters on gas phase volatile inorganic

compounds in mainstream cigarette smoke; Tob. Control 17

i10–i16

Ramage L, Jones A C and Whelan C J 2006 Induction of apoptosis

with tobacco smoke and related products in A549 lung epithelial

cells in vitro; J. Infl amm. 3 3

Rao J K, Letada P, Haverstick D M, Herman M M and Savory J

1998 Modifi cations to the in situ TUNEL method for detection

of apoptosis in paraffi n-embedded tissue sections; Ann. Clin.

Lab Sci. 28 131–137

Robbesom A A, Versteeg E M M, Veercamp J H, van Krieken J H

J M, Bulten H J, Smits H T J, Willems L N A, van Herwaarden

C L A et al. 2003 Morphological quantifi cation of emphysema

in small human lung specimens: comparison of methods and

relation with clinical data; Mod. Pathol. 16 1–7

Saetta M, Shiner R J, Angus G E, Kim W D, Wang N, King

M, Ghezzo H and Cosio M G 1985 Destructive index: a

measurement of lung parenchymal destruction in smokers; Am.

Rev. Respir. Dis. 131 764–769

Schraufstatter I U, Revak S D and Cochrane C G 1984 Protease

and oxidants in experimental pulmonary infl ammatory injury; J.

Clin. Invest. 73 1175–1184

Sherman C B 1991 Health effect of cigarette smoking; Clin. Chest

Med. 12 643

Shah P K and Helfant R H 1988 Smoking and coronary artery

diseases; Chest 94 449–452

Slama K 2008 Global perspective on tobacco control. Part I. The

global state of the tobacco epidemic; Int. J. Tuberc. Lung Dis.

12 3–7

Snider G L, Lucey E C and Stone P J 1985 Animal models of

emphysema; Am. Rev. Respir. Dis. 133 149–169



Stewart B W and Kleihues P (eds) 2003 World cancer report 2003

(Lyon, France: International Agency for Research on Cancer)

pp 21–28

Thornberry N A and Lazebnik Y 1998 Caspases enemies within;

Science 281 1312–1316

Tsujimoto Y 1998 Role of Bcl-2 family proteins in apoptosis:

apoptosomes or mitochondria? Genes Cells 3 697–707

Tuder R M, Petrache I, Elias J A, Voelkel N F and Henson P M

2003 Apoptosis and emphysema: the missing link; Am. J.

Respir. Cell Mol. Biol. 28 551–554

US Department of Health and Human Services 1988 The health

consequences of smoking: nicotine addiction: a report of the

Surgeon General (Rockville, MD: Center for Health Promotion

and Education, Offi ce on Smoking and Health) DHHS

Publication No (CDC) 88-8406

US Department of Health and Human Services 1998 Reducing the

health consequences of smoking: 25 years of progress. A report

of the Surgeon General (Rockville, MD: US Department of

Health, Centers for Disease Control, Center for Chronic Disease

Prevention and Health Promotion, Offi ce on Smoking and

Health) DHHS Publication No (CDC) 89-8411

Wald N J and Hackshaw A K 1996 Cigarette smoking: an

epidemiological overview; Br. Med. Bull. 52 3–11

Whiteside G, Cougnon N, Hunt S P and Munglani R 1998

An improved method for detection of apoptosis in

tissue sections and cell culture, using the TUNEL technique

combined with Hoechst stain; Brain Res. Brain Res. Protoc. 2

160–164

Whiteside G and Munglani R 1998 TUNEL, Hoechst and

immunohistochemistry triple-labeling: an improved method for

detection of apoptosis in tissue sections—an update; Brain Res.

Brain Res. Protoc 3 52–53

Wright J L and Churg A 1995 Smoke-induced emphysema in

guinea pigs is associated with morphometric evidence of

collagen breakdown and repair; Am. J. Physiol. Lung Cell Mol.

Physiol. 268 L17–L20

Wright J L and Churg A 2002 A model of tobacco smoke-

induced airfl ow obstruction in the guinea pig; Chest 121

188S–191S

Wright J L, Farmer S G and Churg A 2002 Synthetic serine elastase

inhibitor reduces cigarette smoke–induced emphysema in

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

Date post:	17-Feb-2022
Category:	Documents
Upload:	others
View:	11 times
Download:	0 times

Activated charcoal ﬁ lter effectively reduces p ...

Documents