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Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel

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The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt(ser473) and p-Akt(thr308)) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2-5 times more particulate pollutants (PM(10) and PM(2.5)), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway.
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Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women
in India who cook with biomass fuel
Sanghita Roychoudhury
, Nandan Kumar Mondal
, Sayali Mukherjee, Anindita Dutta,
Shabana Siddique, Manas Ranjan Ray
Department of Experimental Hematology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata 700 026, India
abstractarticle info
Article history:
Received 11 August 2011
Revised 22 October 2011
Accepted 1 December 2011
Available online 8 December 2011
Indoor air pollution
Airway cells
Oxidative stress
The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the air-
ways has been investigated in 187 pre-menopausal women (median age 34 years) from eastern India who
cooked exclusively with biomass and 155 age-matched control women from same locality who cooked
with cleaner fuel liqueed petroleum gas. Compared with control, Papanicolau-stained sputum samples
showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway
epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt
and p-Akt
) proteins in AEC of biomass users, especially in metaplastic and dysplastic
cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species
(ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative
stress. There were 25 times more particulate pollutants (PM
and PM
), 72% more nitrogen dioxide and
4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-
using households, and high performance liquid chromatography estimated 6-fold rise in the concentration
of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia,
p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that
cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-
mediated activation of Akt signal transduction pathway.
© 2011 Elsevier Inc. All rights reserved.
About half of the world's population relies on unprocessed solid
biomass (wood, dung and crop residues) as the primary source of
domestic energy (Barnes et al., 1994; Reddy et al., 1997). Biomass
accounts for more than 80% of domestic energy in India, China and
Sub-Saharan Africa and nearly 2 billion kilograms of biomass are
burned daily in the developing nations (Holdren and Smith, 2000).
Burning of biomass fuels emits very high levels of smoke containing
a large number of potentially hazardous pollutants that include respi-
rable particulate matters, carbon monoxide, nitrogen dioxides, sulfur
oxides, and a host of organic compounds some of which (e.g. ben-
zo(a)pyrene, benzene and 1,3-butadine) are known human carcino-
gens (Smith, 2000).
Exposure to high level of indoor air pollution (IAP) from biomass
burning during cooking and room heating is regarded as one of
the most important environmental and public health problems in
developing countries especially in women who cook with these
fuels and their young children (Smith, 2002; Smith and Mehta,
2000). The National Ambient Air Quality Standards of the U.S. Envi-
ronmental Protection Agency requires the daily average concentra-
tion of PM
(particulate matter less than 10 μm in diameter) to be
less than 150 μg/m
and annual average to be less than 50 μg/m
(US EPA, 2002). In contrast, a typical 24-hour average concentration
of PM
in biomass-using household in India ranges from 200 to
5000 μg/m
throughout the year, depending on the type of fuel,
stove and housing (Ezzati and Kammen, 2002; Smith, 1993). Indoor
levels of suspected and known carcinogenic compounds which are
usually present adsorbed on the surface of particulate matter were
found signicantly higher in wood burning homes (Gustafson et al.,
2007, 2008). Wood burners had 50% more personal exposure to 1,3-
butadiene, and indoor levels of benzene and benzo(a)pyrene (BaP)
were 25 times higher in wood-burning homes (Gustafson et al.,
2007, 2008). The median indoor BaP level in the wood-burning
homes of Sweden was 5 times higher than the Swedish health-
based guideline of 0.1 ng/m
(Gustafson et al., 2008). This implies
that a lady who cooks with biomass inhales several times more air-
borne carcinogens than a person who cooks with cleaner fuel. Cook-
ing itself is a signicant risk factor for adenocarcinoma of the lung
Toxicology and Applied Pharmacology 259 (2012) 4553
Corresponding author. Fax: + 91 33 2475 7606.
E-mail address: (M.R. Ray).
Contributed equally to this work.
0041-008X/$ see front matter © 2011 Elsevier Inc. All rights reserved.
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(Ger et al., 1993). Despite these reports, the effect of chronic biomass
smoke exposure on the risk of lung cancer among Indian women is
largely unknown except for a study in northern India that showed
higher incidence of lung cancer among biomass users (Behera and
Balamugesh, 2005). Moreover, the underlying mechanism of lung
cancer in association with biomass smoke exposure is currently
In epithelial carcinogenesis, progressive accumulation of molecu-
lar lesions, such as activation of oncogenes, inactivation of tumor
suppressor genes and methylation of promoter is paralleled by a
morphological journey of the cells from normalcy to malignancy
via progressively more severe abnormalities: metaplasiadysplasia
carcinoma-in-situ-invasive cancer. Metaplasia is thus the initial mor-
phological change in this journey towards neoplasia (Grubb, 1988).
Squamous metaplasia usually develops as an adaptive response to
toxic insults and the cells behave differently from that of normal air-
way epithelium. Metaplasia of squamous epithelial cells is an indicator
of predisposition to lung cancer (Djuricic and Plamenac, 1999).
Akt or protein kinase B (PKB) is a serine/threonine kinase.
Active form of this protein controls key cellular processes like glu-
cose metabolism, cell cycle progression, apoptosis, and cell survival
(Brunet et al., 1999; Burow et al., 2000; Eves et al., 1998; Kulik
and Weber, 1998; Madrid et al., 2000; Monick et al., 2001a,
2001b). Activation of Akt requires its translocation to the plasma
membrane and it's binding with phosphatidylinositol triphosphate
via pleckstrin homology domain. Phosphorylation of Akt at thr
by phosphoinositide-dependent kinase 1 partially activates Akt
(Alessi et al., 1996), and its full activation is achieved by additional
phosphorylation at ser
by phosphoinositide dependent kinase 2
(Toker and Newton, 2000). Following activation, Akt detaches itself
from the cell membrane, migrates to the cytoplasm or the nucleus,
and starts mediating pro-survival and anti-apoptotic effects in
part via phosphorylation and inhibition of the Bcl-2 homolog
BAD, phosphorylation and inactivation of FoxO subfamily of fork-
head transcription factors, and transcriptional activation of Yes-
associated protein (Brunet et al., 1999; Datta et al., 1997). Akt pro-
motes tumorigenesis, and this activity is mediated by a variety of
mechanisms that include down-regulation of its inhibitor phospha-
tase and tension homologue deleted on chromosome 10 (PTEN),
activation of the proto-oncogene Ras, and up-regulation of growth
factor receptors (Luo et al., 2003). Change in Akt expression can be
clinically relevant. Up-regulation of phosphorylated Akt (p-Akt) is
considered an early event in the pathway of bronchial (Al-Saad
et al., 2009; Balsara et al., 2004; Chun et al., 2003; Tsao et al.,
2003), oral (Watanabe et al., 2009), and mammary (Al-Bazz et al.,
We have previously demonstrated that chronic biomass exposures
induce oxidative stress (Dutta et al., 2011), chromosomal and DNA
damage (Mondal et al., 2010, 2011a), inappropriate repair of dam-
aged DNA (Mondal et al., 2010) and elevated ribosome biogenesis
(Mondal et al., 2009, 2011b) in airway cells of rural women in India.
These ndings may suggest higher risk of cancer in the airways and
the lung of biomass users. Because metaplasia and dysplasia of airway
epithelial cells are early cellular changes associated with lung cancer
and activated Akt (p-Akt
and p-Akt
) signal transduction
pathway plays an important role in tumorigenesis, we hypothesized
that chronic inhalation of biomass smoke may cause oxidative
stress-induced Akt activation which, in turn, may promote carcino-
genesis in the airways. To test this hypothesis, we examined in this
study the impact of chronic biomass smoke exposure on Akt activa-
tion and dysplastic changes in airway epithelial cells in a group of
never-smoking, premenopausal women from eastern India who
used to cook exclusively with biomass fuel for the past 5 years or
more. We have compared the ndings with an age-matched group
of control women from the same locality who cooked with cleaner
fuel liqueed petroleum gas (LPG).
Materials and methods
Chemicals. Hematoxylin (CAS no. 517-28-2), Orange-G6 (CAS no.
1936-15-8), Eosin Y (CAS no. 17372-87-1), Light green SF yellowish
(CAS no. 5141-20-8), Bismarck brown (CAS no. 10114-58-6), Phos-
photungstic acid (CAS no. 12067-99-1), Pararosaniline (CAS no.
70426-60-7), Phosphoric acid (CAS no. 9066-91-5), Sulfanilamide
(CAS no. 63-74-1), HEPES (CAS no. 7365-45-9), BSA(CAS no. 9048-
46-8), EDTA (CAS no. 60-00-4), Dimethyl formamide (CAS no. 68-
12-2), trans,trans muconic acid (t,t-MA) and 2,7-dichlorouorescein
diacetate (CAS no. 2044-85-1) were purchased from Sigma-Aldrich
Chemicals, Saint Louis, MO, USA. H
(CAS no. 7722-44-1), Potash
alum (CAS no. 7784-24-9), Potassium chloride (CAS no. 7447-40-7),
Mercuric oxide (CAS no. 21908-53-2), Mercuric chloride (CAS no.
7487-94-7), Sodium hydroxide (CAS no. 95077-05-7), Formaldehyde
(CAS no. 98615-67-9) and Acetic Acid (CAS no. 64-19-7) were
obtained from Merck, Mumbai, India. The source of Tris (CAS no.
77-86-1) and Lithium carbonate (CAS no. 554-13-2) was from SISCO
Research Laboratories, India. Methanol (CAS no. 67-56-1) and Ethanol
(CAS no. 64-17-5) were from S.D. Fine-Chem Ltd., Mumbai, India and
Bengal Chemicals & Pharmaceuticals Ltd., Kolkata, India respectively.
All the chemicals were of analytical reagent grade. For immunocyto-
chemistry, primary antibodies like rabbit polyclonal p-Akt
and p-Akt
were purchased from Santa Cruz, CA, USA and
Abcam, Tokyo, Japan respectively. Secondary antibody anti-rabbit
IgG, F(ab)2-HRP, N-(1-naphthyl)-ethylenediamine dihydrochloride
(CAS no. 1465-25-4) and 50× DAB (CAS no. 7411-49-6) were from
Santa Cruz, USA, and phosphatase inhibitor cocktail from Calbiochem,
USA, (Cat no. 524625).
Subjects. A total of 342 pre-menopausal women aged between 28
and 42 years from 8 villages of West Bengal, a state in eastern India,
were enrolled in this study. They attended health check-up camps or-
ganized in different villages with the help of the local administrative
bodies and non-government organizations. Among the participants,
187 women (age 2842 years, median 34 years) cooked daily for
26 h exclusively with wood, cow dung and agricultural refuse, such
as bamboo, jute stick, paddy husk, hay and dried leaves for the past
5 years or more. The remaining 155 women aged 2742 years, medi-
an age 33 years from same locality used to cook with cleaner fuel LPG
and were considered as control.
Inclusion and exclusion criteria. The inclusion criteria were i. appar-
ently healthy, pre-menopausal married women, ii. actively engaged
in daily household cooking for the past 5 years or more, iii. non-
smokers and non-chewers of tobacco, iv. having a body mass
index> 15 and b30 kg/m
. Exclusion criteria were i. users of mixed
fuel (biomass plus LPG/coal/kerosene) ii. using oral contraceptive iii.
had a recent or past history of malignancy, and iv. currently under
Collection of background data. During personal interview with fe-
male members of the research team each participant was asked to
furnish information about age, education, family size and income,
habit, cooking time per day, years of cooking, fuel and oven type,
location of kitchen, health problems in the past 3 months and the
last 1 year. As most of the participants were poorly educated, the
researchers entered their responses in structured questionnaire
forms on their behalf. The Ethics Committee of Chittaranjan National
Cancer Institute approved the study protocol. The research was
conducted according to the principles of the Helsinki Declaration.
Measurement of particulate pollution in indoor air. Particulate mat-
ter (PM) with aerodynamic diameter of less than 10 μm (PM
) and
2.5 μm (PM
) was measured in the cooking areas with a portable,
battery-operated real-time laser photometer (DustTrakAerosol
46 S. Roychoudhury et al. / Toxicology and Applied Pharmacology 259 (2012) 4553
Monitor, model 8520, TSI Inc., MN, USA) that contained 10-mm nylon
Dor-Oliver cyclone and operated at a ow rate of 1.7l/min, measuring
particle load in the concentration range of 1 μg to 100 mg/m
. The
monitor was calibrated to the standard ISO 12103-1 A1 test dust.
Air sampling was done in 8 randomly selected households (4
biomass- and 4 LPG-using) in each of the 8 villages for three consec-
utive days, 8 h/day (7.0015.00 h) covering both cooking and non-
cooking time. Since biomass-using women cook in a sitting position
23 ft away from the open chullah (oven), the monitor was placed
in the breathing zone of the cook, 2.5 ft above oor level on a wooden
stool, 3 ft away from the chullah. LPG users, on the other hand, cook
in a standing position and the monitor was placed accordingly at a
height of 4.5 ft. We used two monitors for simultaneous measure-
ment of PM
and PM
values were reduced by a correction
factor of 2.77 (Siddiqui et al., 2009) while a correction factor of
2.50 was used for PM
(Chung et al., 2001; Lehocky and Williams,
Measurement of SO
and B(a)P in indoor air. Air sampling for
sulfur dioxide (SO
) and nitrogen dioxide (NO
) in indoor air was
done with the help of gaseous impingers attached with the high vol-
ume samplers (Envirotech Instruments, New Delhi, India). SO
measured by modied West and Gaeke (1956) method. In essence,
from air was absorbed in a solution of 0.04 M potassium
tetrachloro-mercurate (10.86 g mercuric chloride, 0.066 g EDTA and
6.0 g potassium chloride in water and the volume made to 1 l, pH
4.0). The resultant dichlorosulphitomercurate complex was made to
react with para-rosaniline and 0.2% formaldehyde to form the in-
tensely colored pararosaniline methylsulphonic acid. The absorbance
of the solution is measured in a spectrophotometer. NO
was mea-
sured by the Jacobs and Hochheiser (1958) method. NO
was collect-
ed in absorbing solution that contained 4.0 g of sodium hydroxide
and 1.0 g of sodium arsenite in 1.0 l of distilled water. Following reac-
tion with 85% phosphoric acid, 2.85% sulfanilamide and 1% aqueous
solution of N-(1-naphthyl)-ethylenediamine di-hydrochloride, nitrite
ion was produced and its concentration was measured spectrophoto-
metrically with the absorbance at 540 nm.
Data on suspended particulate matter (SPM)-laded B(a)P was
obtained from the Central Pollution Control Board, Delhi. B(a)P, col-
lected on glass ber lters and extracted in toluene, was measured
following the procedure of US EPA (TO-13) (US EPA, 1999) using
gas chromatograph (Hewlett Packard, Palo Alto, USA) equipped
with ame ionization detector and Ultra-2 fused silica capillary col-
umn. Hydrogen was used as carrier gas. The injector and detector
temperatures were 300 °C and 320 °C, respectively. The column
oven temperature was programmed at 7 °C/min from 120 °C to
300 °C. The injection volume was 1.5 μl.
Measurement of t,t-MA in urine. Current exposure of benzene was
estimated from the level of benzene metabolite trans,trans-muconic
acid (t,t-MA) in urine by high performance liquid chromatography
with ultraviolet detector (HPLC-UV; Waters, USA) following the
method of Ducos et al. (1990). Urine (2550 ml) was collected within
an hour after completion of cooking for lunch (between 13.00 and
14.00 h) into 100-ml plastic screw-cap vials for analysis of t,t-MA. In
brief, the samples collected on the spot were protected from light,
brought to the laboratory and stored at 20 °C until analysis. The
samples were thawed and a 1 ml aliquot of each was passed through
a Bond elute extraction cartridge lled with 500 mg of SAX sorbent
preconditioned with 3 ml of methanol and 3 ml of distilled water.
The cartridge was washed with 3 ml of 1% acetic acid solution. Then
the t,t-MA was eluted with 3 ml of a 10% aqueous acetic acid. t,t-MA
was measured in HPLC, 10 μl fraction of the elute was used per injec-
tion in column lled with LiChrosorb C18, 5 μM (Waters, USA),
and the detector was set at 259 nm. The eluent was a mixture of 1%
aqueous acetic acid and methanol (9:1, v/v). With a ow rate of
1.2 ml/min, the retention time of t,t-MA was 10 min and the duration
of an analytical run was 20 min. A stock solution of t,t-MA (100 mg/l,
Sigma Chem, USA) was prepared in 10% acetic acid.
Collection of sputum. Early morning sputa of three consecutive days
were collected from each participant in sterile plastic cups (Erkilic
et al., 2003). Three smears were prepared from each freshly collected
sputum samples from the non-transparent highly viscous parts on
clean glass slides and were air dried. One of the slides was xed at
the site of collection with 95% ethanol for Papanicolau (Pap) staining
and other two in cold methanol for immunocytochemical assessment
of Akt expression. Remaining sputum samples were diluted in 20 ml
of phosphate-buffered saline (PBS) containing 0.1% dithiothreitol
(Sigma Chem, USA) in sterile containers and were transferred to the
laboratory in ice box.
Pap staining for cytopathology of airway epithelial cells. Cytology of
exfoliated airway epithelial cells (AEC) present in expectorated
sputa was evaluated after staining the slides with Papanicolau (Pap)
staining following the procedure of Hughes and Dodds (1968). Pap-
stained slides were coded and examined under light microscope
(Leitz, Germany) at 400× and 1000 × magnication. At least 10 high
power elds (hpf, 40× objectives with 10× eyepiece) per slide were
examined. Squamous metaplasia and dysplasia were diagnosed on
the basis of cytological criteria after examining at least 400 epithelial
cells from three Pap-stained slides of each participant. Metaplastic
cells appear as single or loose cluster of bright orange or dark blue-
green-stained cells with glassyappearance. The cell shape is often ir-
regular, size varies between 10 and 25 μm and the chromatin is dense,
greater than normal and nucleoli are seldom seen. The characteristic
features of dysplasia are polymorphism in size and shape of the cell
and the nucleus, markedly increased nuclearcytoplasmic ratio, ab-
normality in chromatin distribution, nuclear membrane indentations
and intensely acidophilic cytoplasm (Grubb, 1988; Naib, 1996).
Specimen adequacy criteria. The sputum samples were cytopa-
thologically classied into six categories following the criteria of
Neumann et al. (2009): category I. nondiagnostic, insufcient
pulmonary material; category II. nondiagnostic, distorted, poorly pre-
served or stained cells; category III. benign, normal cells are present;
category IV. atypical cells are present, probably benign; category V.
atypical cells are present, suspicion of malignancy; and category VI.
malignant cells present. When a sputum slide contained at least 50
alveolar macrophages and grouped in category III or above, it was
considered adequate specimen of the lower airways (Neumann
et al., 2009).
Assessment of Akt activation by immunocytochemistry (ICC). Two ac-
tive forms of phosphorylated Akt (phosphorylation at serine 473,
and phosphorylation at threonine 308, p-Akt
were detected in AEC by ICC. In brief, methanol-xed sputum slides
were washed thrice with ice-cold PBS, incubated with 1:100 dilu-
tion of phosphatase inhibitor cocktail containing ve inhibitors (im-
idazole, sodium uoride, sodium molybdate, sodium orthovanadate
and sodium tartrate) for 5 min at 4 °C, and blocked in 3% BSA
(Sigma-Aldrich Chemicals, Saint Louis, MO, USA) for 1 h at room
temperature following overnight incubation in darkness with Rabbit
polyclonal p-Akt
(Santa Cruz, USA) and p-Akt
UK) primary antibodies (diluted 1:50 and 1:200 in 1% BSA respec-
tively) in a humid box at 4 °C. After washing with PBS, slides were
incubated with anti-rabbit IgG, F(ab)2-HRP (Santa Cruz., USA) sec-
ondary antibody diluted 1:500 in 1% BSA for 90 min. The slides were
developed by incubating with substrate for HRP for 45 min in dark-
ness followed by washing with distilled water and counterstaining
with hematoxylin, dehydration in graded ethanol and mounted in
distyrene plasticizer xylene (DPX).
47S. Roychoudhury et al. / Toxicology and Applied Pharmacology 259 (2012) 4553
Flow cytometric measurement of ROS. Reactive oxygen species (ROS)
that includes superoxide radical, hydrogen peroxide, hydroxyl radical
and singlet oxygen are continuously generated from mitochondrial
respiratory chain and have powerful oxidative potential. ROS are
known to be capable of resulting in the oxidative damages. We used
6-carboxy-2,7-dichlorodihydrouorescein diacetate (DCFH-DA) for
measurement of ROS generation in sputum cells employing ow
cytometry (Rothe and Valet, 1990). The sputum samples collected in
PBS containing 0.1% dithiothreitol were centrifuged at 2500 rpm for
10 min to collect the cell pellet. DCFH-DA working solution (10 μM,)
was added to the cell suspension, and then incubated at 37 °C for
15 min. Cells were then washed once, resuspended in PBS and kept
on ice for an immediate detection by a ow cytometer (FACS Calibur
with sorter, Becton Dickinson [BD], San Jose, CA, USA) with Cell Quest
software (BD, USA). DCFH-DA is cleaved intracellularly by nonspecic
esterases to form DCFH, which is further oxidized by ROS generated
by respiratory burst forming the uorescent compound DCF (Smith
and Weidemann, 1993). Green uorescence by DCF was recorded in
uorescence channel-1 and was expressed as mean uorescence inten-
sity (MFI) in arbitrary unit.
Spectrophotometric measurement of SOD. Sputum cell suspensions
(1.0 ml) were mixed with 500 μl of lysis buffer [0.05 M Tris (pH 7.4),
0.15 M NaCl, 1% Nonidet P-40, with added protease and phosphatase
inhibitors: 1 protease minitab (Roche Biochemicals, Indianapolis,
IN)/10 ml and 1X phosphatase inhibitor mixture (no. 524625;
Calbiochem)] followed by four 20 s pulses of sonication. Samples
were kept in ice in between the sonication pulses. The activity of
the antioxidant enzyme SOD was assayed in whole sputum extracts
spectrophotometrically following the procedure of Paoletti et al.
(1986). Presence of SOD in sample was reected by proportionate
inhibition of the rate of NADH oxidation. This was calculated after
measuring the absorbance of the samples and standard at 340 nm in
a UV-spectrophotometer (Shimadzu, Japan) at 1 min intervals up to
5 min. The absorbance (OD) values were graphically plotted against
time after mercaptoethanol addition (at 0, 1, 2, 3, 4, and 5 min). SOD
activity in lysates was calculated from the standard curve and was
expressed as U/ml.
Statistical analysis. Results are presented as mean±standard devi-
ation (SD) or median with range. Analysis between groups was per-
formed using Student's t-test, Chi-square test, or MannWhitney U
test, as applicable. The possibility of an association between measured
parameters with age, BMI, family income, education, cooking hours
per day, lifetime duration of cooking (cooking-years), kitchen loca-
tion, family size, number of smokers in family, use of mosquito repel-
lant, and PM
and PM
levels in cooking areas was tested using
univariate regression analysis. Variables that showed signicant asso-
ciation were later included in a backward stepwise multiple regres-
sion model to adjust for their effects. Statistical analyses were
performed using EPI info 6 and SPSS statistical software (Statistical
Package for Social Sciences for Windows, release 10.0, SPSS Inc.,
Chicago, USA) and p b.05 was considered signicant.
Demographic and socio-economic characteristics of study popula-
tion are summarized in Table 1. The LPG and biomass using women
were comparable with respect to age, body mass index, cooking
time per day and food habit. However, they differed with respect
to education and family income which were signicantly lower
among biomass users (p b0.05). Moreover, a substantial number of
biomass-using households did not possess separate kitchen (p b0.05
compared with control) and they used to cook in a space adjacent
to living room.
Particulate pollutants in indoor air
The mean concentration± SD of particulate pollutants in indoor
air is presented in Fig. 1. It clearly shows that the levels of PM
and PM
in cooking areas were signicantly higher (p b0.001)
in BMF-using than that of LPG-using kitchen both during cooking
(5- and 4-times respectively) and non-cooking (2.5- and 2-times
respectively) hours.
and B(a)P levels in indoor air
The concentration of SO
in indoor air of cooking areas of biomass-
using homes was 4.6± 1.1 μg/m
compared with 3.9± 1.6 μg/m
LPG-using homes, and the difference was not signicant (p>0.05).
The concentration of NO
, however, was signicantly higher in cook-
ing areas of biomass-using homes (17.6±4.2 vs. 10.2 ± 3.4 μg/m
pb0.05). Similarly, SPM-laden B(a)P in indoor air of biomass-using
homes was 4-times more than LPG-using homes (4.1 ± 1.8 vs. 1.1 ±
0.4 ng/m
Concentration of t,t-MA in urine
The concentration of t,t-MA, a benzene metabolite and a biomark-
er of benzene exposure, was measured in urine by HPLC. In contrast
to 1.1± 0.7 mg/l t,t-MA in control women who cooked with LPG, bio-
mass users had 6.4±4.3 mg/l t,t-MA in urine (p b0.001 in Student's
t-test). In control women, the range of urinary t,t-MA was 0.3
2.9 mg/l with a median of 0.9 mg/l, whereas biomass using women
had a range of 0.920.5 mg/l with a median of 5.1 mg/l (pb0.0001
in MannWhitney U-test). The results suggest remarkably higher
benzene exposure among biomass users.
Cellular changes and prevalence of metaplasia and dysplasia in airway
Women who cooked with biomass fuel had a number of cellular
changes in expectorated sputa. Compared with control, sputa of
biomass users had increased number of total cells (86.5±22.3 [SD]
vs. 49.4± 8.5 cells/high power eld [hpf] in control, p b0.0001),
neutrophils (66.4±10.3 vs. 41.5 ± 6.8/hpf, p b0.0001), eosinophils
(1.6 ± 0.4 vs. 0 .2 ± 0.1/hpf, p b0.0001), lymphocyte (5.5± 1.0 vs. 3.8±
0.7, p b0.05), alveolar macrophages (7.1± 1.7 vs. 1.6 ± 0.6/hpf,
Table 1
Demographic and socio-economic characteristics of biomass and LPG-using women.
Variable LPG-users
Age in year, median (range) 33 (2742) 34 (2842) NS
Median body mass index (kg/m
) 24.4 24.1 NS
Cooking years, median (range) 14 (525) 15 (526) NS
Cooking hours per day, median (range) 3 (25) 3 (26) NS
Years of schooling, median (range) 7 (214) 2 (010) b0.05
Homes with separate kitchen (%) 81.9 57.8 b0.05
Smoker in family (%) 43.9 44.9 NS
Use of mosquito repellant at home (%) 74.2 72.2 NS
Food habit (%)
Vegetarian 3.2 3.7 NS
Mixed 96.8 96.3 NS
Members in family, median (range) 4 (26) 4 (27) NS
Mean (±SD) family income per month (in US $) 9 4± 22 46 ± 14 b0.05
The LPG and biomass fuel users were compared by Chi-square test (for results
presented as percentages), MannWhitney U test (for median values with range)
and Student's t-test (for mean ±SD) and p b0.05 was considered signicant; n, number
of subjects; NS, statistically not signicant.
48 S. Roychoudhury et al. / Toxicology and Applied Pharmacology 259 (2012) 4553
pb0.0001) and basal, parabasal and columnar epithelial cells (5.9± 1.7
vs. 2.3± 0.8/hpf, pb0.0001).
Morphological features of squamous metaplasia were present in
sputa of 40 out of 187 (21.4%) biomass-using women in contrast to
10 of 155 (6.4%) control women. Moreover, dysplasia of epithelial
cells was found in 8/187 (4.3%) biomass-using women compared
with 1/155 (0.6%) of control (Table 2). Thus, metaplasia and dysplasia
of airway cells were 3.3- and 7.2-times more frequent, respectively in
women who cooked with biomass compared with their neighbors
who used LPG as cooking fuel (p b0.001 in Chi-square test).
Kitchen location had appreciable inuence on the cytopathologi-
cal changes in airway cells. For example, women from biomass-
using households that lacked separate kitchen (cooking is done in a
space adjacent to the living room; in most cases they are poorest of
the poor and had a single room per family) had greater prevalence
of squamous metaplasia and dysplasia than those having a separate
kitchen. However, the differences in cytological changes between
women cooking in a separate or adjacent kitchen were not statistical-
ly signicant (p=0.595 for metaplasia and p = 0.467 for dysplasia).
Years of cooking also inuenced airway cytology. The prevalence
of metaplasia and dysplasia was more in women who were cooking
with biomass for the past 15 years or more than those with
514 years' cooking experience. The difference in the prevalence of
dysplasia (p=0.033), but not that of metaplasia (p = 0.0625), be-
tween these two groups was statistically signicant. Among LPG
users also, women with 15 years of cooking experience had greater
prevalence of squamous metaplasia (p = 0.136) and dysplasia
(p= 0.0) than those with shorter exposure-years (Table 2).
Expression of p-Akt in normal, metaplastic and dysplastic AEC
In ICC, expression p-Akt
was conned to the nucleus whereas
expression was both nuclear and cytoplasmic in AEC of
biomass as well as LPG-using women (Fig. 2). Three point two
percent of cytologically normal AEC of control women expressed
while 8.9% of normal AEC of biomass-using women
expressed p-Akt
(Fig. 3). On the other hand, p-Akt
expressed in 9.9% and 32.4% of cytologically normal AEC of control
and biomass-using women, respectively. Expression of p-Akt
and p-Akt
increased dramatically when the cells were trans-
formed from normalcy to metaplasia and dysplasia. Fig. 3 shows
that 56% and 62% of metaplastic AEC and 82% and 86% of dysplastic
AEC in control and biomass users respectively expressed p-Akt
Similarly, p-Akt
expressions were recorded in 81% and 83% of
metaplastic cells and 92% and 91% of dysplastic epithelial cells of
LPG and biomass users, respectively.
Generation of ROS and SOD level of sputum
The MFI of DCFH-DA was 718.6 ± 175.9 in sputum cells obtained
from biomass-using women compared with 356.5 ± 88.3 of LPG
users (pb0.001), implying a 2-fold increase in ROS generation in spu-
tum cells of biomass users. In contrast, the level of SOD in sputum was
41% lower than the control (549.3 ± 112.2 vs. 776.4 ± 427.7 U/ml,
pb0.001). Collectively, the results suggest oxidative stress among
biomass users. ROS generation was positively associated with PM
(OR= 1.45, 95% CI 1.192.58) and PM
(OR= 1.66, 95%CI 1.23
2.88) levels in indoor air and t,t-MA excretion in urine (OR= 1.37,
95%CI 1.182.06).
Association between particulate pollutants and biomarkers for effects
In univariate analysis, PM
and PM
and SPM-laden B(a)P
levels in indoor air, t,t-MA level in urine, lower family income,
lower education, and adjacent kitchen were positively associated
with the prevalence of squamous metaplasia, dysplasia, p-Akt expres-
sion and ROS generation in airway cells. After controlling confound-
ing factors in multivariate logistic regression, a strong positive
association was observed between cellular changes, p-Akt expression
and PM
, B(a)P and t,t-MA levels, especially the concen-
tration of SPM-laden B(a)P (Table 3).
Squamous metaplasia in the airways usually develops as an adap-
tive response to toxic insults, and metaplasia often leads to dysplasia
when cell turnover becomes more rapid (Snead et al., 2003). The pre-
sent study has shown that women who cook with unprocessed solid
biomass had signicantly higher prevalence of these changes in air-
way epithelial cells, implying higher risk of cancer in the airways
Fig. 1. Comparison of particulate air pollution in indoor air of biomass and LPG-using kitchen. The levels of both PM
and PM
(in micrograms per cubic meter of air) were re-
markably higher in biomass-using kitchen during cooking (a) as well as non-cooking (b) time. Bars represent standard deviation of mean values. *pb0.001 in Student's t test.
Table 2
Prevalence of metaplasia and dysplasia of airway epithelial cells in biomass- and LPG-
using women.
Subjects Metaplasia
Biomass-using women, overall (n = 187) 21.4 4.3
With separate kitchen (n = 108) 20.4 3.7
With adjacent kitchen (n = 79) 22.8 5.1
With 15 years of cooking experience (n =105) 24.8 5.7
With 514 years of cooking experience (n =82) 17.1 2.4
LPG-using women, overall (n = 155) 6.4 0.6
With15 years of cooking experience (n =90) 7.8 1.1
With 514 years cooking experience (n =65) 4.6 0
All the data were statistically signicant in Chi-square test.
49S. Roychoudhury et al. / Toxicology and Applied Pharmacology 259 (2012) 4553
and the lung (Djuricic and Plamenac, 1999; Kamei et al., 1993; Vine
et al., 1990). In addition to carcinogenesis, squamous metaplasia of
airway epithelial cells is an indicator of predisposition to an abnormal
forced expiratory volume in one second/forced vital capacity ratio
and consequent airway obstruction that may lead to chronic obstruc-
tive pulmonary disease (COPD) (Madison et al., 1984). Therefore,
increased prevalence of squamous metaplasia among rural women
who used to cook with biomass suggests that these women had a
greater risk of developing lung diseases including COPD and cancer.
Another important nding of this study is up-regulated p-Akt ex-
pression by airway cells of biomass users. Activation of Akt was not
restricted to cytologically abnormal cells as p-Akt was detectable in
cytologically normal AEC also. This is not unexpected, because p-Akt
expression has been reported in 27.3% of normal human bronchial bi-
opsy specimens (Tsao et al., 2003). However, p-Akt expression was
especially increased in metaplastic and dysplastic cells irrespective
of whether the sample was collected from biomass-using or control
women. In agreement with the present observation, Chun et al.
(2003) have found upregulation of p-Akt in pre-malignant bronchial
epithelial cells compared with normal cells. Moreover, elevated
p-Akt activity has been demonstrated in metaplastic and dysplastic
areas of the bronchial epithelium of patients with lung cancer, where-
as normal and hyperplastic bronchial epithelia of these patients
exhibited little or no activity (Balsara et al., 2004). In another study,
p-Akt expression was recorded in 4488% of metaplastic and dysplas-
tic cells whereas only 33% of non-small cell lung cancer specimens
expressed p-Akt (Tsao et al., 2003). Likewise, Watanabe et al.
(2009) found p-Akt-expression in dysplastic and cancer tissues but
not in normal human tongue tissue. Collectively, these reports sug-
gest activation of Akt signaling pathway as an early event in oral
and lung carcinogenesis (Balsara et al., 2004; Tsao et al., 2003).
We found a positive association between particulate pollution,
and B(a)P in indoor air, and t,t-MA excretion in urine and Akt
activation. It may suggest that activation of Akt signal transduction
pathway among biomass users was mediated by, among others, gas-
eous pollutant nitrogen dioxide, respirable particulate matter with
Fig. 2. Immunocytochemical localization of phosphorylated Akt (p-Akt
in a and b and p-Akt
in c and d) in metaplastic (a,c) and dysplastic (b,d) airway epithelial cells in
sputum of never-smoking pre-menopausal women who used to cook exclusively with wood, dung and crop residues. Magnication × 1000.
Fig. 3. Histograms showing percentage of exfoliated normal, metaplastic and dysplastic airway epithelial cells in sputum expressing phosphorylated Akt
and p-Akt
LPG- and biomass-using women. Bars represent standard deviation of mean.*p b0.0001 compared with LPG users in Student's t test.
50 S. Roychoudhury et al. / Toxicology and Applied Pharmacology 259 (2012) 4553
adsorbed carcinogenic polycyclic hydrocarbon B(a)P on their surface,
and benzene which are abundant in biomass smoke (Wafula et al.,
1990; Zhang and Smith, 1996). Consistent with this, Akt activation
has been reported following exposures to environmental carcinogens
(West et al., 2003). Alternatively, biomass smoke can activate Akt
via generation of oxidative stress, which is known to activate Akt
(Esposito et al., 2003). This appears to be a possibility because deple-
tion of antioxidant enzymes and enhancement of lipid peroxidation
have been reported in Turkish women who cooked with biomass
fuels (Gani et al., 2000). In the present study also, we found rise in
ROS and concomitant depletion of SOD, suggesting oxidative stress
among biomass users. Even short-term exposure to biomass smoke
has been shown to reduce plasma antioxidant levels in laboratory
animals (Sezer et al., 2006), and squamous metaplasia has been
observed in rat larynx following chronic exposure to wood smoke
for 90 days (Tesfaigzi et al., 2002). Oxidative stress in the lungs causes
reduction in the activity of histone deacetylase-2 and elevation in
the Akt signaling pathway leading to inammatory response in the
lungs (Marwick and Chung, 2010). In agreement with this, we
found airway inammation in biomass-using women in terms of
increased numbers of neutrophils, eosinophils, lymphocytes and
alveolar macrophages. We have recently shown elevated levels of
pro-inammatory cytokine TNF-αand neutrophil chemoattractant
interleukin-8 in circulation of biomass-using women (Banerjee
et al., in press). Inammation can partially account for the up-
regulation of phosphorylated Akt in airway cells of biomass users, be-
cause TNF-αstimulates the phosphorylation of Akt (Lee et al., 2010)
in order to up-regulate Akt signaling-mediated expression of intercel-
lular adhesion molecule-1 and vascular cell adhesion molecule-1 in
human lung epithelial cells (Oh and Kwon, 2009). Besides, cytokines
including TNF-αinduce matrix metalloproteinase-9, and contribute
to airway inammation (Lee et al., 2010). Increased expression of
in airway cells has been documented in chromium-
induced pulmonary inammation in mice (Beaver et al., 2009). Rhi-
novirus infection in mice causes neutrophilic and lymphocytic airway
inammation and the action is mediated by phosphorylation of Akt in
airway cells (Newcomb et al., 2008). In fact, Akt activation in airway
cells seemed to be an early event of lung inammation. For instance,
cigarette smoke extract causes lung inammation via Akt activation-
mediated cyclooxygenase-2 and prostaglandin E
production (Yang
et al., 2009).
There are some limitations of this study. First, we did not monitor
personal exposures to particulate pollutants. Although we measured
particulate matter, SPM-laden B(a)P, SO
and NO
in indoor air and
benzene metabolite in urine, it is possible that Akt activation is a re-
sult of the action of hundreds of other organic compounds present
in biomass smoke. Second, besides cooking fuel (biomass), cooking
oil fumes can induce Akt. For instance, a study in Taiwan has shown
that cooking oil fumes induce p-Akt, two anti-apoptosis proteins-
inhibitor of apoptosis protein 2 (IAP2) and survivin, and inhibit
pro-apoptotic caspase 3 leading to lung cancer development among
Chinese women (Hung et al., 2007). Cooking oil fume exposure can
explain in part the observed prevalence of metaplasia and dysplasia
among control women of this study who cooked regularly with LPG.
Third, thyroid hormone triiodothyronine (T3) induces a rapid in-
crease in Akt phosphorylation and increased NO production in a
dose-dependent manner (Carrillo-Sepúlveda et al., 2010). Although
no participating woman of this study reported thyroid ailment and
we did not recruit women who were currently under medication
still we cannot rule out the possibility of underlying thyroid disease,
because medical facility and women's understanding about health
in the study areas were poor (Jonas et al., 2010). Despite these limita-
tions, however, the general pattern of the results between biomass
and LPG users was consistent and largely different.
In essence, this study has shown that cooking with biomass
increases the risk of metaplastic and dysplastic changes and up-
regulated expression of Akt in poor, relatively young housewives in
rural India. Consistent with this, chronic exposures to biomass
smoke have been implicated as a major risk factor for lung cancer
among women in India (Behera and Balamugesh, 2005; Smith,
2000; Smith and Mehta, 2003). Currently, lung cancer is the 5th lead-
ing site of cancer among non-smoking women in eastern India where
the study has been conducted, and the affected women are over-
whelmingly non-smokers (Nandakumar et al., 2004). Environmental
factors including exposure to air pollution are believed to be largely
responsible for this, as abnormal columnar epithelial cells and squa-
mous metaplasia in the sputum of young adults and children have
been attributed to air pollution exposure (Plamenac et al., 1973,
1978). On the basis of the present ndings, efforts should be made
to reduce indoor air pollution from biomass burning substantially
through immediate (by increasing kitchen ventilation) and long-
term (switching to cleaner energy options) air pollution abatement
measures, and p-Akt may be investigated as a potential target in
future lung cancer prevention studies. Indeed, pharmacological inhi-
bition of Akt phosphorylation has been shown to inhibit premalig-
nant and malignant growth in human bronchial epithelium (Chun
et al., 2003).
Conict of interest statement
The authors declare that there are no conicts of interest.
The authors gratefully acknowledge the nancial support received
from Central Pollution Control Board, Government of India, Delhi, in
carrying out this study.
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Multivariate logistic regression analysis for association between particulate pollution in indoor air and urinary benzene metabolite with cellular changes in the airways after con-
trolling potential confounders.
Particulate pollutants in indoor air t,t-MA in urine
OR 95% CI OR 95% CI OR 95% CI OR 95% CI
Metaplasia 1.35 1.141.72 1.49 1.202.76 1.23 1.021.58 1.65 1.282.98
Dysplasia 1.47 1.212.05 1.87 1.473.15 1.30 1.041.92 2.02 1.473.77
1.33 1.081.82 1.61 1.332.74 1.22 1.071.62 1.44 1.232.62
1.39 1.141.83 1.48 1.201.92 1.21 1.031.49 1.57 1.332.90
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... Prevalence of mucus plugs, goblet cell hyperplasia, and nuclear anomaly of columnar epithelial cells was found to be higher in urban subjects exposed to high levels of urban air pollution in Kolkata as compared to controls drawn from relatively cleaner peri-urban zones (Ray and Lahiri 2010). However, as compared to these controls, Papanicolau-stained sputum samples of biomass users showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cells (AEC) (Roychoudhury et al. 2012). Siderophages (ironcontaining macrophages in sputum indicative of either past intrathoracic bleeding or extravasations of red blood cells into the alveoli due to a sluggish blood flow) were abundant in sputum of the residents of Delhi and Kolkata implying microscopic hemorrhage in the lungs (Roy et al. 2001). ...
... Elevated levels of elastase (a proteolytic enzyme found in the lysosomes of neutrophils and alveolar macrophages capable of destroying elastin and causing alveolar degradation) were reported in urban populations of Delhi and Kolkata with some of the highest levels recorded in automobile service station workers, traffic policemen and roadside hawkers . Cumulative exposure to biomass smoke has also been shown to increase oxidative stress-mediated activation of Akt signal transduction potentially increasing the risk of lung cancer (Roychoudhury et al. 2012). Currently, the biological evidence of activated carcinogenic mechanisms associated with air pollution is substantial and growing, and is corroborated by studies conducted in the Indian context. ...
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Air pollution ranks among the leading risk factors contributing to the burden of disease in South Asia with household and ambient air pollution accounting for 6 percent and 3 percent respectively of the total national burden of disease in India. Both urban and rural communities bear this burden in terms of premature mortality and disability-adjusted life years, resulting from excess risks of communicable and non-communicable diseases. We review the information pertaining to exposures to fine particulate matter and air toxics together with the attributable disease burden estimates. We also provide a summary of the results from recent assessments on carcinogenicity of ambient and household air pollution conducted by The International Agency for Research on Cancer. We conclude with a list of specific priorities for action related to air toxics and cancer in India.
... Biomass smoke also activates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway that controls key cellular processes such as glucose metabolism, cell cycle progression, apoptosis and inflammation. Cooking with biomass fuel upregulated the expression of active (phosphorylated) p-Akt ser473 and p-Akt thr308 in bronchial epithelial cells, especially in metaplastic and dysplastic cells, alveolar macrophages, airway neutrophils and peripheral blood lymphocytes of rural women in India in their child-bearing age [215,216]. ...
... Biomass smoke exposures increases the risk of multinucleation, metaplasia and dysplasia of bronchial epithelial cells in women (Fig. 14.3; [215,216,54]). Metaplasia and dysplasia of airway cells are the initial morphological changes in the journey towards neoplasia [227]. ...
Indoor air pollution (IAP) due to daily household cooking with unprocessed solid biomass such as wood, dung and crop residues is a serious health hazard in the poor, developing countries of Asia, sub-Saharan Africa and Latin America. Globally, 2.8 billion people use biomass for domestic energy. Incomplete combustion of biomass emits smoke that contains a host of potentially health-damaging particulate and gaseous pollutants, some of which like benzo(a)pyrene, 1,3-butadiene and benzene are known human carcinogens. IAP from biomass burning is responsible for excess mortality and morbidity. An estimated four million deaths, mostly from cardio-pulmonary causes, have been attributed to biomass use. Children, women and the elderly people are most vulnerable. Chronic inhalation of biomass smoke induces lung function decrement, increases the risk of life-threatening chronic obstructive pulmonary disease, evokes pulmonary and systemic inflammation and consequent oxidative stress, and contributes to the development of hypertension and cardio-vascular diseases. Daily household cooking with biomass was associated with higher incidences of anemia, platelet hyperactivity, and altered number and activities of the immune cells. Oxidative stress generated by biomass smoke mediates chromosomal and DNA damage and impairment in DNA repair mechanism in the exposed cells. In addition, chronic inhalation of biomass smoke up-regulates protein kinase B/Akt signaling and metaplasia and dysplasia of airway cells, implying increased risk of lung cancer. Women who cooked with biomass also had altered serotonergic activity with greater prevalence of depression. Thus, IAP due to household cooking with biomass adversely affects both physical and mental health of the people.
... In this context, studies on the carcinogenic mechanisms carried out in the airway cells of women exposed to chronic inhalation of biomass smoke in India, documented a higher concentration at home of PM 2.5 and PM 10 particles, nitrogen dioxide, and benzo (a) pyrenes, and higher levels of benzene metabolites in the urine than women that use clean fuels in the same country. It is presumed that chronic inhalation of the aforementioned compounds leads to an increase in oxidative stress in the cells of the respiratory epithelium, which in turn, leads to a higher frequency of metaplasia and dysplasia of airway epithelial cells (Mondal et al., 2010(Mondal et al., , 2015Roychoudhury et al., 2012;Mukherjee et al., 2013). It should be noted that combustion of wood might be a necessary factor for the potential carcinogenic effect of wood smoke; this statement is supported by the results that Matrat et al. (2019), reported in 2019, in which wood dust exposure was not associated with an increased risk of developing lung cancer. ...
Introduction: Exposure to biomass combustion products, particularly firewood, has been considered as a potential carcinogen for developing lung cancer. In this regard, current evidence is widely heterogeneous; besides, in most studies, wood smoke exposure is not appropriately quantified, which further complicates the analysis of wood smoke as a potential carcinogen. The aim of the present study was to estimate the risk of developing lung cancer according to the degree of exposure to wood smoke in patients who use firewood for cooking. Material and methods: We performed a case-control study that included 482 patients with lung cancer (cases) and 592 hospital controls. Exposure to wood smoke was evaluated as a dichotomous variable (i.e. yes or no); in patients with prior wood smoke exposure, an index of exposure in hours per year was calculated (WSEI). Using bivariate and multivariate logistic regression analyses, the odds ratio (OR) between wood smoke exposure and lung cancer were calculated. Results: The ORs for developing lung cancer (raw and adjusted) for a WSEI > 100 h/year were OR 1.55 [95% confidence interval (CI), 1.06-2.26) and OR 2.26 (95% CI, 1.50-3.40), respectively; the ORs (raw and adjusted) for WSEI >300 h/year were OR 1.76 (95% CI, 1.06-2.91) and OR 3.19 (95% CI, 1.83-5.55), respectively. Conclusions: Exposure to wood smoke is a risk factor for lung cancer; furthermore, this effect maintains a dose-response relationship which has a multiplicative effect with smoking.
... One study has shown that chronic exposure to biomass smoke causes morphological changes to the epithelial compartment consistent with emphysema and the small airway damage elicited by cigarette smoke [18]. In line with this, another group has shown that the epithelium can undergo metaplastic and dysplastic changes in response to chronic exposure to the pollutants in biomass smoke [19]. However, more information is needed to further our understanding of how the smoke generated by the combustion of forested regions, which has a different composition to other forms of environmental smoke, impacts the viability and function of the immunological barrier imparted by the airway epithelium. ...
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Background Individuals with respiratory disease are being increasingly exposed to wildfire smoke as populations encroach further into forested regions and climate change continues to bring higher temperatures with lower rainfall. Frequent exposures have significant potential to accelerate conditions such as chronic obstructive pulmonary disease (COPD) which is characterised by an exaggerated inflammatory response to environmental stimuli. Here we employ models of human airway epithelium exposed to wildfire smoke-extract (WFSE) to examine modulation in airway epithelial cell (AEC) survival, fragility and barrier function. Methods Submerged cultures of small airway epithelial cells (SAEC) and differentiated air-liquid interface (ALI) cultures of primary bronchial AEC (bAEC) were treated for 1–24 h with 1–10% WFSE generated from plant species found in the Australian bushland. Autophagy (LC3-II and Sequestosome), apoptosis (Poly-(ADP)-Ribose Polymerase (PARP) cleavage) and tight junction proteins were measured using western blot. Barrier function was assessed via permeability of fluorescein tracers and measuring trans-epithelial electrical resistance. The production of IL-6 was assessed using ELISA. Results Primary epithelial models exposed to WFSE exhibited a significant blockade in autophagy as evidenced by an increase in LC3-II coupled with a concomitant elevation in Sequestosome abundance. These exposures also induced significant PARP cleavage indicative of apoptotic changes. ALI cultures of bAEC treated with 5% WFSE demonstrated barrier dysfunction with significant increases in paracellular molecular permeability and ionic conductance, and a reduction in the abundance of the tight junction proteins ZO-1 and Claudin-1. These cultures also exhibited increased IL-6 secretion consistent with the aberrant and pro-inflammatory repair response observed in the COPD airways. Further, blocks in autophagy and barrier disruption were significantly elevated in response to WFSE in comparison to similar exposures with cigarette smoke-extract. Conclusion WFSE inhibits autophagic flux and induces barrier dysfunction in the airway epithelium. As autophagy is a central regulator of cellular repair, viability, and inflammation, targeting the block in autophagic flux may ameliorate the consequences of wildfire smoke-exposure for individuals with pre-existing respiratory conditions. Electronic supplementary material The online version of this article (10.1186/s12931-018-0945-2) contains supplementary material, which is available to authorized users.
... In addition, a limited number of Indian studies corroborate the broader global evidence for pathophysiological effects that may underlie the development of chronic non-communicable respiratory and cardiovascular disease. These Indian studies report findings that air pollution has been associated with a range of underlying effects, including cytopathological changes (such as increased prevalence of mucus plugs and siderophages, goblet cell hyperplasia, and nuclear anomalies of columnar epithelial cells), airway inflammation (indicated by increased counts of neutrophils, lymphocytes, eosinophils, and alveolar macrophages, and by higher sputum levels of IL-6, IL-8, and TNF-␣) and oxidative stress (indicated by enhanced ROS generation and depletion of SOD activity) Dutta et al. 2012;Lahiri et al. 2008;Ray et al. 2006;Roy et al. 2001;Roychoudhury et al. 2012). ...
... The organic fraction of particulate matter containing PAH metabolites had enhanced the cell proliferation through interaction with two major signalling pathways such as epidermal growth factor receptor pathway and the serine/threonine kinase Akt pathway [3]. Roy Choudhury [4] reported that women, in India, using biomass for cooking had up regulation of phosphorylated Akt proteins in airway epithelial cells, showing the risk of carcinogenesis via oxidative stress-mediated activation of the Akt signal transduction pathway as compared to women using liquefied petroleum gas (LPG). Burning of coal come out to be a stronger risk factor for lung cancer than wood burning [3,[5][6][7] 3 billion people still cook and heat their homes using open fires, stoves burning biomass (wood, animal dung and crop waste) and coal. ...
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p> Objective: Aim of the study was to screen the suspended particulate matter for their role in enhancing angiogenesis employing crown gall tumor assay. Methods: Particulate matter (PM<sub>10</sub>) samples were collected, on glass fiber filter paper at the rate of 1.12–1.14 m<sup>3/</sup>min for 8 hr using high volume sampler, from six different sites of Amritsar city. Aqueous extracts of suspended particulate matter were prepared using a mechanical shaker for 24 hour and the filtrate was centrifuged and lyophilized. Results: Maximum (34.67±1.764) tumors were induced by samples collected from Crystal chowk whereas tumor inducing ratio (TIR) was found to be comparably high for two sites viz., Crystal chowk and Bhandari bridge. Conclusion: High tumor inducing ratio at the above-mentioned sites was correlated to high traffic emission from automobile exhaust. Crown gall tumor assay has been proved to be rapid, economical and reliable screening assay for angiogenesis agent.</p
... Earlier, we have shown that ethanolic leaf extract of Thuja occidentalis and isolated flavonols from it exert apoptotic effect on a kras mutated NSCLC cell line (A549) sparing the normal embryonic lung cell (L-132) [14,15] , but further studies to isolate different flavonol fractions and identify if any particular flavonol fraction had greater anti-cancer potential against NSCLC cell line A549 or any particular fraction acted more strongly to ameliorate BaP induced lung toxicity are lacking. Therefore, the hypotheses to be tested in this study were: i) If different flavonol-rich fractions could be isolated, identified and tested for their individual ability to induce cytotoxicity in lung cancer cells, A549 in vitro; ii) If any particular fragment could show greater ability to induce apoptosis, and whether this fragment could be effective in reducing BaP induced lung toxicity in the mice model as well, indicating its possible chemotherapeutic use; and iii) if the underlying mechanism of drug induced redox state modulation and the signal transduction, in particular, could be understood to know if it was mediated through the modulation of Akt signalling pathway, a major pathway involved in initiation of BaP induced toxicity as reported by earlier workers [16,17] . ...
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Relative anticancer potentials of six flavonol fractions (F1-F6) isolated from Thuja occidentalis extract were first evaluated against lung cancer cells A549 in vitro, and fraction- F4 showing the maximum protective ability, was then tested in mice intoxicated with Benzo(a)pyrene (BaP), a known carcinogen with major effect on lung, to learn if this could also have ameliorative action against lung toxicity and tissue damage in mice in vivo. Chemical nature of F1-F6 fractions was confirmed with specific flavonol confirmatory test and mass spectral analysis; all fractions were tested for their possible anticancer effects against A549 cells. Results suggested that fraction 4 (F4) had the strongest anticancer effect. When treated to BaP intoxicated mice, F4 induced recovery of damaged lung tissue, presumably through inhibition of ROS generation, and enhanced production of major antioxidant molecules, that in turn blocked PI3K-activated expressions of Akt. Decrease in Bcl2/Bax ratio, over-expression of p53 gene and activation of caspase 3 were observed in tissues of F4 treated mice, further confirming apoptotic cell death as its major target. The F4 fraction of Thuja occidentalis extract showed remarkable apoptotic potential against lung cancer cells and ameliorative ability against BaP induced lung toxicity.
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The study was carried out to examine whether chronic exposure to smoke during daily household cooking with biomass fuel (BMF) elicits changes in airway cytology and expressions of Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2]), Keap1 (Kelch-like erythroid-cell-derived protein with CNC homology [ECH]-associated protein 1), and NQO1 (NAD(P)H:quinone oxidoreductase 1) proteins in the airways. For this, 282 BMF-using women (median age 34 year) and 236 age-matched women who cooked with liquefied petroleum gas (LPG) were enrolled. Particulate matter with diameters of < 10 µm (PM10) and < 2.5 µm (PM2.5) were measured in indoor air with real-time laser photometer. Routine hematology, sputum cytology, Nrf2, Keap1, NQO1, and generation of reactive oxygen species (ROS) along with the levels of superoxide dismutase (SOD) and catalase were measured in both groups. PM10 and PM2.5 levels were significantly higher in BMF-using households compared to LPG. Compared with LPG users, BMF users had 32% more leukocytes in circulation and their sputa were 1.4-times more cellular with significant increase in absolute number of neutrophils, lymphocytes, eosinophils, and alveolar macrophages, suggesting airway inflammation. ROS generation was 1.5-times higher in blood neutrophils and 34% higher in sputum cells of BMF users while erythrocyte SOD was 31% lower and plasma catalase was relatively unchanged, suggesting oxidative stress. In BMF users, Keap1 expression was reduced, the percentage of AEC with nuclear expression of Nrf2 was two- to three-times more, and NQO1 level in sputum cell lysate was two-times higher than that of LPG users. In conclusion, cooking with BMF was associated with Nrf2 activation and elevated NQO1 protein level in the airways. The changes may be adaptive cellular response to counteract biomass smoke-elicited oxidative stress and inflammation-related tissue injury in the airways.
Exposure to particulate emissions poses a variety of public health concerns worldwide, specifically in developing countries. This review summarized the documented studies on indoor particulate matter (PM) emissions and their major health concerns in South Asia. Reviewed literature illustrated the alarming levels of indoor air pollution (IAP) in India, Pakistan, Nepal, and Bangladesh, while Sri Lanka and Bhutan are confronted with relatively lower levels, albeit not safe. To our knowledge, data on this issue are absent from Afghanistan and Maldives. We found that the reported levels of PM10 and PM2.5 in Nepal, Pakistan, Bangladesh, and India were 2-65, 3-30, 4-22, 2-28 and 1-139, 2-180, 3-77, 1-40 fold higher than WHO standards for indoor PM10 (50 μg/m(3)) and PM2.5 (25 μg/m(3)), respectively. Regarding IAP-mediated health concerns, mortality rates and incidences of respiratory and non-respiratory diseases were increasing with alarming rates, specifically in India, Pakistan, Nepal, and Bangladesh. The major cause might be the reliance of approximately 80% population on conventional biomass burning in the region. Current review also highlighted the prospects of IAP reduction strategies, which in future can help to improve the status of indoor air quality and public health in South Asia.
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Three types of respirable dust samplers were positioned side-by-side as area samplers within three coal-fired electric power generating facilities. Respirable dust readings were taken using two direct-reading aerosol monitors (MIE PDM-3 Miniram and the TSI Model 8520 DustTrak) and the results compared to side-by-side respirable coal dust concentrations. Both direct-reading instruments use optical sensors for detecting dust concentrations, and in this study the air was passed through a 10-mm cyclone prior to detection. Respirable samples were collected using a 10-mm cyclone with a 5-µm PVC filter connected to a constant flow pump calibrated at 1.7 L/min. The samples were collected for fifteen 8-hr shifts over a one-month period. Respirable dust concentrations ranged from 0.23 to 10.83 mg/m3. The responses of each of the direct-reading instruments were compared to the respirable values. Neither of the two direct-reading instruments provided values that were identical to each other or the respirable samplers, but regression analyses indicated high coefficient of determination (R2) values (0.85 and 0.94). Other statistical methods (analysis of variance, pair wise t-tests, and mixed effect models) found no significant differences (p>0.05) between the data sets for the direct-reading instruments and the respirable samples. It was concluded that the two direct-reading instruments can be used to measure respirable coal dust.
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Hundreds of millions of people rely on woodfuels for most of their energy needs, despite the problems associated with traditional use of woodfuels. Modern, efficient biomass stoves can alleviate some of these problems by reducing some householders' cash outlays for fuel, diminishing the time others must spend to collect fuel, reducing air pollution, and relieving local pressure on wood resources. The study explores the successes and failures of stove programs and suggests how adoption rates can be improved more consistently. Under the right conditions, the social, economic, and environmental benefits of promoting improved stoves are large. Programs must be targeted carefully, however, to situations in which people pay high prices for fuel or walk long distances to collect fuelwood or other biomass materials. Subsidies may aid in the distribution of stoves but may not result in actual stove use. Ultimately, dissemination programs are most effective when they allow for interaction and feedback between stove designers, producers, and users.
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OBJECTIVE: To compare the sputum smear cytology and cell block methods for specimen adequacy, cytologic quality and diagnostic accuracy in the diagnosis of lung cancer. STUDY DESIGN: We assessed 2,524 sputum specimens from 768 patients. The specimens were prepared as smears and cell blocks for cy-topathologic examination between March 1, 1992, and December 31, 1998. The smear and cell block slides were evaluated both separately and together, and the results were compared with radiologic and histopathologic diagnoses. RESULTS: The sensitivity of the smear method was 69.4% and specificity was 99.5%. The sensitivity of the cell block method was 84.4% and specificity, 100%. The sensitivity of the smear and cell block together was 87.6% and specificity, 99.5%. CONCLUSION: The cell block method increases the sensitivity and specificity of sputum cytology, and when smear and cell block slides are evaluated together, sensitivity reaches its highest value. Therefore, application of smear and cell block methods together seems most useful in the diagnosis of lung cancer. Carcinoma of the lung is the leading cause of cancer mortality in the western world. 1 Sputum cytology complements radiology in the diagnostic workup of lung cancer and can be useful in the identification of lung carcino-ma, especially at early or occult stages. 2,3 This method has sensitivity and specificity of 64.5% and 99.7%, respectively. 2 Sputum cytology also has the advantage of being easily applicable and noninva-sive and is cost effective. 4 Although sputum cytol-ogy has been performed routinely as smear cytol-ogy, the cell block method, which is not routinely When smear and cell block specimens are evaluated together, the sensitivity rate for detection of carcinoma is higher.
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early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphory- lation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32 P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of b-catenin, resulting in nuclear accumulation and transcriptional activity of b-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear b-catenin in human alveolar macrophages and expression of genes that require nuclear b-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of b-catenin in the nucleus of any cell, including alveolar macrophages. The Journal of Immunology, 2001, 166: 4713- 4720.
To evaluate the effects of cigarette smoking and biomass (dried dung) smoke on the oxidant–antioxidant status, three groups each with 5 rabbits were used. Groups of rabbits were exposed to either cigarette smoke, dried dung smoke or dry air, 1 h daily for one month. Protein carbonyls, prostaglandin F2α and malondialdehyde levels were significantly increased and protein sulfhydryls levels were significantly decreased in the cigarette smoke group compared with the control group. Only protein sulfhydryls levels were significantly decreased in dung group compared with the control group. Short course exposure to both cigarette smoke and biomass smoke decreased plasma antioxidant levels but only cigarette smoke increased plasma oxidant levels, whereas biomass smoke did not produce any change.
This review examines developing-country air pollution from the standpoint of a useful analysis technique that has been under development in recent years: "total exposure assessment'. The review is composed of four parts: a brief description of the historical and current relationship between energy use and air pollution; an explanation of the idea of exposure assessment and the power that it can bring to analyses of the health impacts of air pollution; focusing on developing countries, a global exposure assessment; and a review of the health effects literature relevant to the micro-environments found to harbor the largest human exposures. -from Author
Nitrogen dioxide is determined in air in the presence of much higher concentrations of sulfur dioxide on an hourly basis by using an automatic 24-hour sampler. Air is aspirated through a fritted-glass bubbler containing 0.1N alkali solution. The absorbed nitrogen dioxide is determined colorimetrically as the azo dye by using it to diazotize sulfanilamide in phosphoric acid and then coupling it with N-(1-naphthyl)-ethylenediamine dihydrochloride. Nitrogen dioxide in the order of parts per hundred million of air can be determined. The sulfur dioxide present is also absorbed, but when oxidized to sulfate with hydrogen peroxide, does not interfere with the reaction.
Sulfur dioxide in the atmosphere is removed and concentrated by scrubbing through 0.1M sodium tetrachloromercurate(II). Stable, nonvolatile disulfitomercurate(II) is formed. The subsequent determination of the isolated sulfur dioxide is based on the red-violet color produced when p-rosaniline hydrochloride-hydrochloric acid mixture (0.04% dye - 6% concentrated acid) and formaldehyde (0.2%) are added to the sampling solution. The absorption maximum is at 560 mμ and the color is temperature-independent and stable for several hours. The method is sensitive (0.005 to 0.2 p.p.m. with a 38.2-liter air sample scrubbed through 10.0 ml. of sampling solution) and should be useful in the absolute determination of sulfur dioxide in air pollution surveys. Nitrogen dioxide is the only common interference.