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Toxicologic Pathology
http://tpx.sagepub.com/content/38/7/1085
The online version of this article can be found at:
DOI: 10.1177/0192623310385139
2010 38: 1085 originally published online 5 October 2010Toxicol Pathol
Nandan K. Mondal, Amrita Roy, Bidisha Mukherjee, Debangshu Das and Manas R. Ray
Lymphocytes : A Study among Premenopausal Women in Rural India
Indoor Air Pollution from Biomass Burning Activates Akt in Airway Cells and Peripheral Blood
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Indoor Air Pollution from Biomass Burning Activates Akt in Airway
Cells and Peripheral Blood Lymphocytes: A Study among
Premenopausal Women in Rural India
NANDAN K. MONDAL
1
,AMRITA ROY
2
,BIDISHA MUKHERJEE
1
,DEBANGSHU DAS
1
,AND MANAS R. RAY
1
1
Department of Experimental Hematology, Chittaranjan National Cancer Institute, Kolkata, India
2
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata, India
ABSTRACT
Biomass burning is a major source of indoor air pollution in rural India. The authors investigated in this study whether cumulative exposures to
biomass smoke cause activation of the serine/threonine kinase Akt in airway cells and peripheral blood lymphocytes (PBL). For this, the authors
enrolled 87 premenopausal (median age 34 years), nonsmoking women who used to cook with biomass (wood, dung, crop wastes) and 85 age-
matched control women who cooked with cleaner fuel liquefied petroleum gas. Immunocytochemical and immunoblotting assays revealed signif-
icantly higher levels of phosphorylated forms of Akt protein (p-Akt
ser473
and p-Akt
thr308
) in PBL, airway epithelial cells, alveolar macrophages, and
neutrophils in sputum of biomass-using women than control. Akt activation in biomass users was associated with marked rise in generation of reac-
tive oxygen species and concomitant depletion of superoxide dismutase. Measurement of particulate matter having a diameter of less than 10 and
2.5 mm in indoor air by real-time aerosol monitor showed 2 to 4 times more particulate pollution in biomass-using households, and Akt activation was
positively associated with particulate pollution after controlling potential confounders. The findings suggest that chronic exposure to biomass smoke
activates Akt, possibly via generation of oxidative stress.
Keywords: biomass fuel; airway cells; lymphocytes; p-Akt; premenopausal women; India.
INTRODUCTION
Akt or protein kinase B (PKB) is a serine/threonine kinase.
It controls key cellular processes like glucose metabolism, cell
cycle progression, apoptosis, and cell survival (Monick, Carter,
et al. 2001; Brunet et al. 1999; Burow et al. 2000; Eves et al.
1998; Kulik and Weber 1998; Madrid et al. 2000; Monick,
Mallampalli, et al. 2001). Activation of Akt requires its
translocation to the plasma membrane and binding with
phosphatidylinositol triphosphate (PIP3) via pleckstrin
homology (PH) domain. Phosphorylation of Akt at Thr
308
by
phosphoinositide-dependent kinase 1 (PDK1) partially acti-
vates Akt (Alessi et al. 1996), and its full activation is achieved
by additional phosphorylation at Ser
473
by phosphoinositide-
dependent kinase 2 (PDK2) (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 (Datta et al. 1997; Brunet et al. 1999).
In addition, Akt mediates cell proliferation and migration via
the phosphorylation and activation of the endothelial nitric
oxide synthase and glycogen synthase kinase-3b, which is
functional during cell cycle progression, and inactivation of
p
21CIP1
and p
27KIP1
, which are inhibitors of cyclin-dependent
kinases (Fosbrink et al. 2006; Diehl et al. 1998). Akt activation
facilitates cell survival even after DNA adducts formation
(West et al. 2003). In conformity with this, excessive activation
of Akt has been implicated in the development of a wide range
of human cancers (Scheid and Woodgett 2001). Akt mediates
its tumor-promoting activity by a variety of mechanisms that
include down-regulation of its inhibitor phosphatase and ten-
sion homologue deleted on chromosome 10 (PTEN), activation
of the proto-oncogene Ras, and up-regulation of growth factor
receptors (Luo, Manning, and Cantley 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 (Tsao et al. 2003;
Chun et al. 2003; Balsara et al. 2004; Al-Saad et al. 2009), oral
(Watanabe et al. 2009), and mammary (Al-Bazz et al. 2009)
carcinogenesis. Besides, Akt activation may cause relaxation
of vascular smooth muscle cells via increased nitric oxide
Address for Correspondence: Manas R. Ray, PhD, Department of
Experimental Hematology, Chittaranjan National Cancer Institute, 37,
S.P. Mukherjee Road, Kolkata-700 026, India; phone: þ91-33-2476 5101; fax:
þ91-33-2475 7606; e-mail: manasrray@rediffmail.com.
Abbreviations: IAP, indoor air pollution; PM, particulate matter; BMF,
biomass fuel; LPG, liquefied petroleum gas; AEC, airway epithelial cells;
AM, alveolar macrophages; PBL, peripheral blood lymphocytes; PKB,
protein kinase B; PI3K, phosphatidylinositol 3-kinase; PIP3,
phosphatidylinositol triphosphate; PDK1, phosphoinositide-dependent kinase
1; PTEN, phosphatase-and-tension-homologue-deleted-on-chromosome-10;
ROS, reactive oxygen species; SOD, superoxide dismutase; PAP, Papanicolaou;
EDTA, ethidium diamine tetra acetic acid; PBS, phosphate buffered saline;
BSA, bovine serum albumin; HRP, horseradish peroxidase; SDS–PAGE,
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DCF-DA,
dichlorofluorescein diacetate; MFI, mean fluorescence intensity.
1085
Toxicologic Pathology, 38: 1085-1098, 2010
Copyright #2010 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623310385139
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production (Carrillo-Sepu
´lveda et al. 2010), suppression of
gastric acid secretion (Rotte et al. 2010), airway inflammation
via activation of pro-inflammatory mediators (Beaver et al.
2009; Lee et al. 2010), and perpetuation of inflammation by
delaying neutrophil apoptosis (Salamone et al. 2010).
A large majority of rural women of the developing world
including India are chronically exposed to high level of indoor
air pollution (IAP) for daily household cooking with traditional
biomass fuel (BMF) such as dried cow dung cake, firewood,
and agricultural wastes. Smoke emitted from burning biomass
contains a wide spectrum of pollutants that include carbon
monoxide; coarse, fine, and ultrafine particulate matters; oxi-
des of nitrogen and sulphur; formaldehyde; transitional metals;
volatile organic compounds including benzene; and polycyclic
aromatic hydrocarbons such as benzo(a)pyrene (Zhang and
Smith 1996). Some of these pollutants like benzo(a)pyrene and
benzene are established human carcinogens (Smith 2000).
Women who used to cook with BMF in poorly ventilated
kitchen for 2 to 6 hours a day are believed to be inhaling car-
cinogens equivalent to smoking two packs of cigarettes per day
(Pandey et al. 1989 ). It is reasonable to assume therefore that
chronic exposures to biomass smoke may impair cellular func-
tions, especially in those cells that are at the direct route of
exposure. Given the importance of Akt in regulating cell sur-
vival, growth, and apoptosis, it thus seems important to study
its activity in relation to biomass smoke exposures. To our
knowledge, no such study has been carried out so far. Against
this background, we examined in this study the impact of IAP
from BMF use on Akt activation in exfoliated AEC and inflam-
matory cells present in expectorated sputum as well as in PBL in
a group of never-smoking, premenopausal women from eastern
India who used to cook exclusively with BMF for the past 5
years or more. We have compared the findings with an age-
matched group of control women from the same neighborhood
who cooked with cleaner fuel liquefied petroleum gas (LPG).
MATERIALS AND METHODS
Subjects
A total of 172 premenopausal women aged between 28 and
42 years from rural areas of West Bengal, a state in eastern
India, were enrolled in this study after obtaining written
informed consent. They attended health checkup camps orga-
nized in different villages with the active cooperation of the
local administrative bodies and nongovernmental organiza-
tions. Among the participants, 87 women (aged 28–42 yr, med-
ian 34 yr) were cooking daily for 3–6 hours 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. Accordingly they were grouped as biomass-users. The
remaining 85 women, aged 27–42 yr, median age 33 yr, were
from the same villages, but they used to cook with cleaner fuel
LPG and were considered as control. Some households, espe-
cially those using biomass as cooking fuel, lacked a separate
kitchen. Women of these households used to cook in a space
adjacent to the living room.
Inclusion and Exclusion Criteria
The inclusioncriteria were apparently healthy, premenopausal
married women actively engaged in household cooking for
the past 5 years or more who were nonsmokers and nonchewers
of tobacco and had a body mass index > 15 and < 30 kg/m
2
.
Women using oral contraceptive, had a history of malignancy,
or were currently under medication were excluded.
During personal interview with female members of the
research team, each participant was requested to furnish infor-
mation about age, education, family size and income, habit,
cooking time per day, years of cooking, fuel and oven type,
ventilation and location of kitchen, and general health prob-
lems in past 3 months and past 1 year. As most of the partici-
pants were poorly educated, the researchers recorded 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.
Collection of Blood and Expectorated Sputum
Venous blood (5 ml) was collected in vacutainer tubes
(Becton Dickinson [BD], San Jose, CA, USA) containing
K
2
EDTA as anticoagulant. Blood was collected at a fixed
time of the day (9.30–10.30 hours) to minimize diurnal var-
iation. Early morning expectorated sputa were collected in
sterile plastic cups for 3 consecutive days to harvest airway
cells following the procedure of Erkilic et al. (2003). Three
smears were prepared from the nontransparent highly viscous
part of each freshly collected sputum sample on clean glass
slides from each day’s sample—one for Papanicolaou (Pap)
staining and two for immunocytochemistry (ICC), one for
p-Akt
ser473
and another for p-Akt
thr308
. After air drying, one
slide marked for Pap staining was fixed with 95%ethanol
and the other two marked for ICC were fixed in ice-cold
methanol for 20 min at the site of collection. The remaining
part of the expectorated sputum was collected in sterile plas-
tic screw-cap tubes containing 20 ml of phosphate-buffered
saline (PBS) with 0.1%dithiotheritol.
PAP Staining for Cytopathology of Airway Cells
Ethanol-fixed slides were brought to the laboratory and
were stained with Papanicolaou (Pap) staining method follow-
ing the procedure of Hughes and Dodds (1968). The slides were
coded and examined under light microscope (Leitz, Germany)
at 400x and 1,000x magnification. At least 10 high power fields
(hpf; 40x objective and 10x eyepiece) per slide were examined.
Sputum cytology and differential distribution of non-squamous
AEC, AM, and inflammatory cells were performed following
the established criteria (Grubb 1988). The slides were coded
and cytological examinations were carried out blindly. The
3-day average value of each parameter was taken as the
representative data of each participant.
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Cytopathological Diagnosis and Specimen Adequacy
Criteria
The sputa were cytopathologically diagnosed into six cate-
gories following the criteria of Neumann et al. (2009): category
I. nondiagnostic, insufficient pulmonary material; category II.
nondiagnostic, distorted, poorly preserved or stained cells;
category III. benign, normal cells are present; category IV. aty-
pical 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 was grouped in category III or
above, it was considered an adequate specimen of the lower air-
ways (Neumann et al. 2009). Squamous metaplasia and dyspla-
sia were diagnosed on the basis of cytological criteria (Grubb
1988; Naib 1996). Metaplastic cells appear as a single or loose
cluster of bright orange or dark blue-green-stained cells with
‘‘glassy’’ appearance. The cell shape is often irregular, size var-
ies between 10–25mm and the chromatin is dense, greater than
normal, and nucleoli are seldom seen. The characteristic fea-
tures of dysplasia are polymorphism in size and shape of the
cell and the nucleus, markedly increased nuclear-cytoplasmic
ratio, abnormality in chromatin distribution, nuclear membrane
indentations, and intensely acidophilic cytoplasm.
Lymphocyte Separation from Peripheral Blood
PBL were separated from EDTA-anticoagulated whole
blood by centrifugation in density gradient (Histopaque
1
-
1077, Sigma Chem, St. Louis, MO, USA) for 10 min at 400
xgat4
C. The lymphocytes were collected in microfuge tubes,
resuspended in 500 ml of ice-cold phosphate buffered saline
(PBS). Smears of lymphocytes were prepared on clean glass
slides for immunocytochemistry (ICC) for localization of phos-
phorylated Akt (p-Akt) protein.
Localization of p-Akt by ICC
Immunocytochemistry of PBL and sputum slides were done
following a standard procedure (Mondal et al. 2010). In brief,
slides containing PBL and sputum cells were air dried and fixed
in ice-cold methanol for 30 min, air dried, washed in PBS
thrice, and blocked in 3%BSA (Sigma–Aldrich Chemicals,
Saint Louis, MO, USA) for 1 hr at room temperature. The
slides were overnight incubated in darkness with rabbit poly-
clonal p-Akt
ser473
(Santa Cruz, CA, USA) and p-Akt
thr308
(Abcam, Tokyo, Japan) primary antibodies (diluted 1:50 and
1:200 in 1%BSA, respectively) in a humid box at 4C. After
washing with PBS, slides were incubated with anti-rabbit IgG,
F(ab’)2-HRP (Santacruz, USA) secondary antibody diluted
1:500 in 1%BSA for 90 min. The slides were developed by
incubating with substrate for HRP for 45 min in darkness fol-
lowed by washing with distilled water and counterstaining with
hematoxylin, dehydration in graded ethanol, and mounted in
distrene plasticizer xylene (DPX). The slides were coded and
examined blindly. For each participant, one sample of PBL and
three samples of sputum obtained from 3 consecutive days were
examined. The 3-day average values of p-Akt
ser473
and p-
Akt
thr308
expressions in sputum cells were taken as the represen-
tative data of each participant.
Cell Lysate Preparation for Immunoblot and SOD Assay
Whole sputum collected in PBS with 0.1%dithiotheritol
was centrifuged for 10 min at 400 x gat 4
0
C. The supernatant
was discarded, and the cells were suspended in fresh PBS and
centrifuged again and the cell pellet was collected. Whole-cell
protein from sputum and PBL were obtained by lysing the cells
on ice with 500 ml 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, Indiana-
polis, IN, USA]/10 ml and 1x phosphatase inhibitor mixture
[no. 524625; Calbiochem, Darmstadt, Germany]) followed by
four 20 s pulses of sonication. Samples were kept in ice in
between the sonication pulses. Lysates were centrifuged at
15,000 x gfor 10 min at 4
0
C in an ependorf microcentrifuge.
The supernatant was collected and used for immunoblotting
(sputum and PBL) and SOD (sputum) assay.
Immunoblot Analysis of p-Akt
Cleared lysates were subjected to SDS–PAGE (10%matrix)
according to the method of Laemmli (1970). Protein concentra-
tions in the lysates were measured using the Bradford assay.
Proteins (60 mg) separated by SDS-PAGE were electrophoreti-
cally transferred to nitrocellulose membrane (ECL; Amersham
Biosciences, Arlington Heights, IL, USA) using a mini trans-
blotter (Bio-Rad, Hercules, CA, USA). Membranes were
blocked, then incubated consecutively with rabbit polyclonal
primary antibodies (1:1,000 dilution) raised against human p-
Akt
ser473
, p-Akt
thr308
, and total Akt (Cell Signaling Technol-
ogy, Beverly, MA, USA) and HRP-conjugated anti-rabbit IgG
secondary antibody (1:2,000 dilution). Immunoreactive proteins
were visualized with enhanced Chemiluminescence method
using luminol reagent kit (Santa Cruz Biotechnology, Santa Cruz,
CA, USA). Protein levels in each band were quantitated using a
Fluor-S scanner and Quantity One software for analysis (Bio-
Rad). The data were analyzed using GraphPad Prism software
(San Diego, CA, USA).
Flow Cytometric Measurement of ROS Generation
Generation of reactive oxygen species (ROS) was measured
in leukocytes (granulocytes, monocytes, and lymphocytes)
present in anticoagulated venous blood and sputum cells by
flow cytometry using DCF-DA following the procedure of
Rothe and Valet (1990). For this, 10,000 events were acquired
in a flow cytometer (FACS Calibur with sorter, Becton
Dickinson [BD], San Jose, CA, USA) using Cell Quest soft-
ware (BD). Respiratory burst and generation of ROS by cells
were associated with emission of green fluorescence that was
recorded in fluorescence channel-1 and was expressed as mean
fluorescence intensity (MFI) in arbitrary unit. In case of blood,
granulocytes, monocytes, and lymphocytes were gated on the
Vol. 38, No. 7, 2010 AKT ACTIVATION AMONG BIOMASS USERS 1087
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basis of their characteristic forward and side scatters on dot plot
and MFI of each population was recorded, while MFI of the
total cells present in sputum was recorded.
Spectrophotometric Measurement of SOD
The activity of the antioxidant enzyme superoxide dismutase
(SOD) was assayed in blood erythrocytes and sputum cell lysate
spectrophotometrically following the procedure of Paoletti et al.
(1986). EDTA anticoagulated blood was centrifuged at 200 x gat
4
0
C for 5 min, the RBC pellet was collected and washed in 0.9%
saline and then lysed with cold distilled water (1: 9, v/v). Follow-
ing centrifugation at 500 x gfor 10 min, the supernatant was
collected. In spectrophotometric cuvette, 800ml of TEA-DEA
buffer containing 100 mM each of triethanolamine (TEA) and
diethanolamine (DEA) (Qualigens, Mumbai, India), 40mlof
7.5 mM nicotinamide adenine dinucleotide reduced disodium
salt (NADH, SRL, Mumbai, India), pH 7.4, 25ml of a mixture
(1:1, v/v) of 0.2 M ethylene diaminetetraacetic acid disodium salt
(Sigma-Aldrich Chem, St. Louis, MO, USA) and 0.1 M manga-
nous chloride (SD-Fine Chem, Mumbai, India), and 100mlof
sample (RBC or sputum cell lysate) were added and mixed well.
The absorbance (OD) was measured at 340 nm in a spectrophot-
ometer (Shimadzu, Kyoto, Japan) immediately (0 min) and 1, 2,
3, 4, and 5 min after addition of mercaptoethanol. SOD activity was
calculated from the standard curve and was expressed as U/ml.
Measurement of PM
10
and PM
2.5
in Indoor Air
Particulate matter with aerodynamic diameter less than 10mm
(PM
10
) and 2.5mm (PM
2.5
) were measured in the cooking areas
with real-time laser photometer (DustTrak
2
Aerosol Monitor,
model 8520, TSI Inc., Shoreview MN, USA) that contained
10-mm nylon Dor-Oliver cyclone and operated at a flow rate
of 1.7 L/min, measuring particle load in the concentration range
of 1mg to 100 mg/m
3
. The monitor was calibrated to the standard
ISO 12103-1 A1 test dust. We used two monitors for simulta-
neous measurement of PM
10
and PM
2.5
. Air sampling was done
in each household for three consecutive days, 8 hours/day (7.00–
15.00 hrs) covering both cooking and noncooking time. The
mean of 3 days was used as the indoor air quality of a single
household. Biomass-using women cook in a sitting position 2–
3 ft away from the open chullah (oven). Accordingly, the monitor
was placed in the breathing zone of the cook 2.5 ft above the floor
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
therefore placed at a height of 4.5 ft. Since laser photometers
make an overestimation of PM levels by 2–3 fold (Lehocky and
Williams 1996), the raw data require reduction using correction
factors. We reduced the 8-hr raw data by dividing with a correc-
tion factor of 2.5 for PM
10
(Siddiqui et al. 2009) and 2.77 for
PM
2.5
(Lehocky and Williams 1996; Chung et al. 2001).
Statistical Analysis
Results are presented as mean +standard deviation (SD)or
median with range. Analysis between groups was performed
using Student’s t-test, chi-square test, or Mann-Whitney Utest,
as applicable. The possibility of an association between
measured parameters with age, BMI, family income, educa-
tion, cooking hours per day, lifetime duration of cooking
(cooking-years), kitchen location, family size, number of
smokers in family, use of mosquito repellant, and PM
10
and
PM
2.5
levels in cooking areas was tested using univariate regres-
sion analysis. Variables that showed significant association were
later included in a backward stepwise multiple regression 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< .05 was considered significant.
RESULTS
Demography
Demographic and socioeconomic characteristics of study
population are summarized in Table 1. The LPG and BMF
users were comparable with respect to age, body mass index,
cooking years, hours of cooking per day, environmental
tobacco smoke for presence of a smoker in the family, food
habit, and use of mosquito repellant. However, BMF users
were less educated (p< .05 in Mann-Whitney Utest), and their
family income was significantly lower than that of their neigh-
bors who used to cook with LPG (p< .05 in Student’s t-test).
Moreover, 42.5%of BMF-using households lacked a separate
kitchen against 17.7%of LPG-using households, and the differ-
ence between these two groups in this regard was significant (p
< .05) in chi-square test.
Particulate Pollutants in Indoor Air
The levels of particulate air pollutants in indoor air in cook-
ing areas were significantly higher (p< .001) in BMF-using
TABLE 1.—Demographic and socioeconomic characteristics of
biomass fuel and LPG-using women.
Variable
LPG users
(n¼85)
Biomass users
(n¼87) p
Age in yr, median (range) 33 (25–42) 34 (24–42) NS
Median body mass index (kg/m
2
) 24.4 24.1 NS
Cooking years, median (range) 14 (5–25) 15 (5–22) NS
Cooking hours per day, median (range) 3 (2–5) 3 (2–6) NS
Years of schooling, median (range) 8 (3–14) 3 (0–10) <.05
Homes with separate kitchen (%) 82.3 57.5 <.05
Smoker in family (%) 43.5 44.8 NS
Use of mosquito repellant at home (%) 74.1 72.4 NS
Food habit, mixed (%) 96.5 96.6 NS
Members in family, median (range) 4 (2–6) 4 (2–7) NS
Family income per month in $US
(mean +SD)
58 +831+7 <.05
The LPG and biomass-using groups were compared by chi-square test (for results
presented as percentages), Mann-Whitney Utest (for median values with range), and
Student’s t-test (for mean +SD) and p< .05 was considered significant; n, number of
subjects; NS, statistically not significant.
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households than that of LPG-using households during cooking
as well as noncooking time (p< .001) (Table 2).
Sputum Cytology
Biomass users had elevated number of total cells in sputum
(89.6 +24.5 vs. 64.1 +11.2 cells/hpf, p< .01) than controls.
Sputa of BMF users contained more AEC (11.9 +3.3 vs. 6.3 +
2.21 cells/hpf, p< .01), neutrophils (57.9 +18.6 vs. 46.6 +
11.8 cells/hpf, p< .01), eosinophils (1.6 +0.8 vs. 0.4 +0.2,
cells/hpf p< .01), lymphocytes (4.5 +1.3 vs. 3.3 +1.1,
cells/hpf p< .05), and AM (12.9 +6.9 vs. 6.7 +3.1 cells/hpf,
p< .01) than that of control. Moreover, AEC metaplasia was
recorded in 20.7%of BMF-using women in contrast to 8.2%
of control, and mild to moderate dysplasia was present in sputa
of 4.5%BMF and 1.2%LPG users (p< .01 in chi-square test).
Expression of p-Akt
ser473
and p-Akt
thr308
Phosphorylated forms of Akt, p-Akt
ser473
, and p-Akt
thr308
were localized by ICC and immunoblotting. Expression of
p-Akt
ser473
was mostly nuclear, while p-Akt
thr308
was
expressed chiefly in the cytoplasm (Figures 1–4). Compared
with control, a statistically significant increase in the percen-
tages of p-Akt
ser473
- and p-Akt
thr308
–expressing epithelial cells,
neutrophils, and AM in sputum and lymphocytes in peripheral
blood were recorded among BMF users (Table 3). BMF users
had 3 times more p-Akt
ser473
-expressing AEC and PBL than
controls. Similarly, they had 4 times more airway neutrophils
and AMs that expressed p-Akt
ser473
when compared with con-
trols. On the other hand, BMF users showed 5-fold increase
over control in the percentage of basal cells of the airways that
expressed p-Akt
thr308
in their cytoplasm. The percentage of
p-Akt
thr308
-expressing cells was 2- to 2.7-fold higher for the
remaining cell types among BMF users (Table 3).
Compared with cytologically normal AEC, the percentage
of p-Akt–expressing cells was remarkably higher (p< .001)
among metaplastic and dysplastic cells both among LPG and
BMF users. Expression of p-Akt
ser473
was found in 56%and
62%of metaplastic cells and 82%and 86%of dysplastic cells
in LPG and BMF users, respectively. Similarly, p-Akt
ser473
expression was recorded in 81%and 83%of metaplastic cells
and 92%and 91%of dysplastic cells in sputa of LPG and BMF
users, respectively.
Controlling age, family income, and kitchen location
as possible confounders, multivariate logistic regression
analysis showed a strong positive association between
percentage of p-Akt
ser473
-expressing AEC with PM
10
(odds
ratio [OR] ¼1.33, 95%confidence interval [95%CI]
1.09–1.62) and PM
2.5
(OR ¼1.45, 95%CI 1.12–1.74) level
in indoor air.
Immunoblotting revealed greater presence of p-Akt in PBL
and sputum cells of women who used to cook with BMF
(Figure 5). Akt phosphorylation positively correlated with
years of cooking with biomass without altering the total Akt
protein level (Figure 5).
ROS and SOD Levels
Flow cytometric analysis showed a significant rise in MFI
DCF-DA among BMF users, suggesting increase in ROS
generation in peripheral blood leukocytes (granulocytes,
monocytes, and lymphocytes) and un-fractionated sputum
cells that contained alveolar macrophages, epithelial
cells, airway neutrophils, eosinophils, and lymphocytes
(Figure 6A). The MFI of DCF-DA was 36%higher in periph-
eral blood granulocytes (572.5 +136.5 vs. 420.6 +143.3 in
control, p< .0001), 25%higher in monocytes (306.4 +
124.8 vs. 245.6 +102.5, p¼.0006), and 14%higher in
lymphocytes (179.4 +91.1 vs. 157.5 +72.7, p¼.0836)
of biomass users, relative to controls. ROS generation was
doubled in sputum cells of biomass users when compared
with that of control (MFI 708.8 +274.9 vs. 346.4 +
188.1, p< .0001). On the other hand, biomass-using women
had 39%and 28%lower levels of SOD in erythrocytes
(156.8 +65.2 vs. 255.1 +59.3 U/ml in control, p<
.0001) and sputum cells (565.3 +115.2 vs. 785.4 +183.8
U/ml, p< .0001), respectively, in comparison with control (Figure
6B).
Controlling age, family income, and position of the kitchen
as possible confounders, multivariate logistic regression analy-
sis indicated a strong positive association between MFI of
DCF-DA and level of PM
10
(OR ¼1.25, 95%CI 1.07–1.45)
and PM
2.5
(OR ¼1.51, 95%CI 1.20–1.93).
ROS, SOD, and Akt Activation
Figure 7 demonstrates progressive rise in the percentages of
p-Akt
ser473
-positive AEC and PBL in relation to years of cook-
ing with BMF. Cooking years correlated positively with gener-
ation of ROS in AEC, in terms of MFI of DCF-DA (Figure 7a),
and negatively with concentration of SOD in erythrocytes (Fig-
ure 7b). Highest percentages of p-Akt
ser473
-positive AEC
(22.7%) and PBL (40.1%) were recorded in women having
longest duration of cooking with biomass (20–24 yrs). Women
in this exposure category showed greatest elevation in ROS
(DCF-DA MFI 743.50) and maximum depletion of erythrocyte
SOD (135 U/ml).
TABLE 2.—Comparison of particulate pollution in indoor air of
cooking areas between LPG- and BMF-using households.
Particulate air pollutant
in cooking areas
LPG-using
households
BMF-using
households
PM
10
(g/m
3
)
Cooking time 129 +42 516 +208*
Noncooking time 72 +21 134 +68*
PM
25
(g/m
3
)
Cooking time 70 +29 269 +118*
Noncooking time 35 +14 72 +41*
Results are expressed as mean +SD.
*p< .001 compared with LPG-using households in Student’s t-test.
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FIGURE 1.—Immunocytochemical localization of phosphorylated forms of Akt protein in AEC present in sputum of biomass- and LPG-using con-
trol women. The cell nuclei were counterstained with hematoxylin. Compared with relatively weak nuclear expression of p-Akt
ser473
in AEC of
control women (A), strong p-Akt
ser473
expression was observed in nuclei (B) as well as in cell membrane (C) of AEC of biomass users. Similarly,
strong p-Akt
thr308
expression was detected in nuclei and cell membrane of AEC of biomass-using women (E,F), while weak cytoplasmic expres-
sion was observed in AEC of control women (D). Magnification x 1,000.
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DISCUSSION
The objective of this study was to examine whether cumu-
lative inhalation of biomass smoke causes activation of the
proto-oncogene Akt in those cells that are at the direct route
of exposure to inhaled pollutant. We studied Akt activity also
in PBL, because biomass smoke contains high concentration
of ultrafine particles (UFPs; particles having aerodynamic dia-
meter of less than 0.1 mm) that can cross the alveolar-capillary
barrier and then circulate throughout the body, causing sys-
temic damage through generation of oxidative stress (Bra¨uner
et al. 2007). We found marked increase in the percentages of
p-Akt
ser473
- and p-Akt
thr308
-positive airway cells and PBL
among BMF users, suggesting up-regulation of Akt activity
in these cells. Akt is one of the key regulators of cell prolifera-
tion and death. It prevents apoptosis, and this effect is mediated
by its influence on downstream targets including forkhead
proteins (Datta et al. 1997), Bcl
XL
(Jones et al. 2000), NF-kB
(Madrid et al. 2000), Bad (Datta et al. 1997), caspase-8 (Jones
et al. 2002), and glycogen synthase kinase (Brazil, Park, and
Hemmings 2002). Transcription factor FOXO 3A exerts
tumor-suppressive functions by inducing transcription of
p
27CIP1
(Di Cristofano et al. 2001); and p-Akt phosphorylates
this transcription factor, causing its functional inactivation and
export from the nucleus (Brunet et al. 1999). Apoptosis of T
and B-lymphocytes were found inhibited in transgenic mice that
overexpressed active Akt (Bommhardt et al. 2004). Conversely,
inhibition of PI3K-Akt signaling pathway induced apoptosis in
rat hepatic stellate cells, even under strong mitotic stimulation
by platelet-derived growth factor (Wang et al. 2008).
The participants of this study were never-smokers and non-
chewers of tobacco. Moreover, exposure to environmental
tobacco smoke for the presence of smoker in the family was
similar among BMF and LPG users. Therefore, greater expres-
sion of p-Akt in BMF-using women cannot be explained by
tobacco use. The villages where the participants resided were
far from the highways and busy road traffic. Bicycle and
cycle rickshaw were the principal mode of transport, and there
were no air-polluting industries within 5 km radius. Thus,
ambient air pollution levels in the study areas seemed
negligible. Besides, the BMF and LPG users were neighbors;
hence the impact of outdoor air pollution was similar in these
two groups. The major difference between these two groups
was significantly higher exposure to particulate air pollution
among BMF users, and this could explain to a large extent the
observed increase in Akt activation. There has been progres-
sive increase in the percentages of p-Akt-expressing cells in
association with increasing cooking years. However, we found
a strong correlation between age and years of cooking with
FIGURE 2.—Immunocytochemical localization of phosphorylated forms of Akt in sputum neutrophils of biomass- and LPG-using control women.
The cell nuclei were counterstained with hematoxylin. A greater proportion of sputum neutrophils of biomass-using women expressed p-Akt
ser473
in nuclei (B) than that of control (A). p-Akt
thr308
expression in general was weak and chiefly cytoplasmic and biomass-users illustrated greater
frequency of positive cells (D) than the control (C). Magnification x 1,000.
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biomass (r¼.0094) or LPG (r¼.0092). Thus, age appears to
be an important contributor to higher p-Akt expression among
women with longer cooking years. In conformity with this,
p-Akt expression has been found significantly higher in color-
ectal carcinoma patients aged 60 years or more when compared
with younger patients (Khor et al. 2004).
Some studies have shown Akt activation in association with
environmental carcinogenesis (West et al. 2003). Since bio-
mass smoke contains a host of mutagens and carcinogens
including benzo(a)pyrene, 1,3-butadiene, and benzene (Wafula
et al. 1990; Zhang and Smith 1996), it is possible that increased
expression of p-Akt was mediated by these mutagens. Alterna-
tively, biomass smoke can activate Akt via generation of oxida-
tive stress, as ROS can activate Akt (Esposito et al. 2003). This
appears to be a distinct possibility as we found rise in ROS and
concomitant depletion of SOD, suggesting oxidative stress
among BMF users. Like the present finding, depletion of anti-
oxidant enzymes and enhancement of lipid peroxidation have
been reported in Turkish women who cooked with BMF (Gani
et al. 2000). Even short-term exposure to biomass smoke has
been shown to reduce plasma antioxidant levels in laboratory
animals (Sezer et al. 2006).
AMs are the first line of cellular defense against inhaled
pollutants in the lung. Human AM plays a critical role in host
defense and in the development of inflammation and fibrosis
in the lung. Proliferation of respiratory epithelium, increase in
the number of AM, emphysematous changes, and bronchoal-
veolar hemorrhage have been observed in rabbits exposed to
dung cake smoke (Fidan et al. 2006). In line with these observa-
tions, we found increased number of AM and inflammatory
cells in expectorated sputum of BMF-using women, suggesting
airway inflammation. AMs have constitutive Akt activation, and
the cells are deficient in PTEN (Flaherty, Monick, and Hinde
2006). This could be a molecular adaptation to survive for pro-
longed periods of time in vivo as the estimated turnover time of
AM in humans is 81 days (Thomas et al. 1976). Akt is involved
in the signal transduction pathway mediating delay of neutrophil
apoptosis induced by inflammatory mediators (Rane and Klein
2009), and its inhibition was found to enhance neutrophil death.
Conversely, neutrophils with increased Akt activity have a pro-
longed lifespan (Zhu et al. 2006). Therefore, Akt activation in
AM and airway neutrophils, as observed in BMF users, may give
these cells survival advantage and perpetuate inflammation. The
net effect could be respiratory impairment. In agreement with
this, BMF users of this study had greater prevalence of upper
(sore throat, sneezing, running nose) and lower respiratory
symptoms (wet and dry cough, wheeze, chest tightness); higher
prevalence of chronic obstructive pulmonary disease (COPD);
FIGURE 3.—Immunocytochemical localization of phosphorylated forms of Akt in AM present in expectorated sputum of biomass- and LPG-using
control women. The cell nuclei were counterstained with hematoxylin. Although the staining intensity of p-Akt
ser473
in AM from control (A) and
biomass-using women (B) is similar, the frequency of positive cells is greater in B than in A. Expression of p-Akt
thr308
, on the other hand, was
weak and chiefly cytoplasmic, and biomass users had greater frequency of p-Akt
thr308
positive AM (D) than that of control (C). Magnification x
1,000.
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and significant decline in pulmonary function especially in
forced vital capacity (FVC), forced expiratory volume in one
second (FEV
1
), FEV
1
/FVC ratio, and mid expiratory flow rate
(FEF
25-75%
) when compared with their neighbors who used to
cook with LPG (data not shown).
The major strengths of the present study are the identified
human cohorts and their focused and detailed examination of
Akt activity in differentiated airway cells and PBLs. However,
the study has its limitations. First, we have only partially
characterized indoor air pollution by measuring PM
10
and
PM
2.5
levels in cooking areas, leaving emission of gaseous pol-
lutants such as oxides of nitrogen and carbon monoxide and
their possible impact on Akt expression undetected. Second,
we have no data on personal exposure of these pollutants.
Third, the study did not attempt to identify potential toxic com-
ponents responsible for these biological changes. For example,
we did not carry out physicochemical analyses of PM
10
and
PM
2.5
such as level of polycyclic aromatic hydrocarbons
FIGURE 4.—Immunocytochemical localization of phosphorylated forms of Akt in PBL of biomass- and LPG-using control women. The cell nuclei
were counterstained with hematoxylin. Expression of p-Akt
Ser473
was strong and both nuclear and cytoplasmic. Biomass-using women had higher
frequency of p-Akt
Ser473
–expressing PBL (B) than control (A). Expression of p-Akt
Thr308
in PBL of control women was negligible (C). In contrast,
strong, cytoplasmic p-Akt
Thr308
expression was found in a large numbers of PBL of biomass users (D). Magnification x 1,000.
TABLE 3.—Changes in nuclear and cytoplasmic expression of two forms of phosphorylated Akt in LPG- and BMF-using women.
Nuclear p-Akt
ser473
expression Cytoplasmic p-Akt
thr308
expression
Cell types LPG users BMF users LPG users BMF users
In expectorated sputum
Parabasal and intermediate epithelial cells (%) 4.3 +1.6 12.4 +4.7** 10.6 +2.8 29.4 +8.2**
Basal epithelial cells (%) 2.1 +1.4 7.4 +2.6** 7.1 +1.5 35.2 +6.2**
Neutrophils (%) 21.8 +6.2 82.5 +16.2** 28.4 +5.9 75.8 +9.9**
Alveolar macrophage (%) 17.1 +5.5 68.8 +8.3** 38.8 +7.2 91.5 +8.4**
In peripheral blood
Lymphocytes (%) 9.0 +3.1 33.8 +9.7** 23.8 +10.8 78.4 +15.7**
The results are expressed as mean percentage of positive cells +standard deviation. An average of 100 basal epithelial cells, 200 parabasal and intermediate epithelial cells, 100
alveolar macrophages, 300 sputum neutrophils, and 500 peripheral blood lymphocytes were examined for each individual.
** p< .001 compared with LPG users in Student’s t-test.
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FIGURE 5.—Western blot analysis of p-Akt
Ser473
(a,c) and p-Akt
Thr308
expression (b,d) in PBL (a,b) and airway cells (c,d) of premenopausal
women who were cooking with LPG or biomass fuel for the past 10–19 (A) and 20–29 (B) years. Whole cell lysates (60g/lane) were used for
immunoblotting. The degree of Akt phosphorylation were quantified by densitometry and normalized with total Akt. The blots and relative inten-
sity of p-Akt
ser473
and p-Akt
the308
proteins shown above them illustrate higher levels of phosphorylated Akt among biomass users. All results are
representative of one of four independent experiments with comparable results.
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including benzo(a)pyrene, elemental carbon, organic carbon,
particle size, and endotoxin content. Endotoxin seems impor-
tant because it elicits inflammation and Akt activation, and
airborne endotoxin concentrations in biomass, burning homes
of Malawi and Nepal have been found significantly higher than
those found in homes in developed countries (Semple et al.
FIGURE 7.—Histograms showing close relationship between years of cooking with BMF, ROS, and SOD levels, and activation (phosphorylation)
of Akt. Increasing cooking years were associated with progressive rise in the percentage of p-Akt
ser473
-expressing AEC (a) and circulating lym-
phocytes (b). The changes were paralleled by increase in ROS generation in airway cells (a) and depletion of SOD in erythrocytes (b). A, 5–9; B,
10–14; C, 15–19; and D, 20–24 years of cooking experience. Bars represent SD.
FIGURE 6.—Histograms showing higher levels of reactive oxygen species (ROS) generation in peripheral blood leukocytes and sputum cells
(A) and lower levels of superoxide dismutase (SOD) enzyme in erythrocytes and sputum cells (B) of biomass-using women in comparison with
LPG-using control. Bars represent standard deviation of mean.
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2010). Fourth, we carried out indoor measurements and
biological sampling for a limited number of days. Therefore,
seasonal variation and climatic changes could have influenced
the measured parameters. However, monitoring of PM was
done in LPG- and biomass-using homes of a village simultane-
ously, as was sampling of sputa and blood. Thus, seasonal
effect does not appear to be a significant confounder. The par-
ticipants of this study who cook regularly were simultaneously
exposed to cooking oil fumes and cooking fuel emissions.
Therefore, up-regulation of Akt can be attributed in part to
cooking-oil fume, as it is capable of inducing up-regulation of
p-Akt in bronchial cells (Hung et al. 2007). However, both LPG
and biomass users of this study were exclusive users of mustard
oil as cooking medium, and the type of cooking and food habit
was similar in these two groups. Therefore, cooking-oil fume
exposure appeared to be similar in both groups, excluding this
as a contributor to Akt activation among BMF users.
In essence, the study has shown up-regulation of Akt
activity in women who used to cook with BMF and are
therefore highly exposed to IAP. p-Akt was detectable in
AEC of control women also. This is consistent with the
report that showed p-Akt expression in 27.3%of normal
human bronchial biopsy specimens (Tsao et al. 2003). Bio-
mass users of this study had significant rise in the preva-
lence of metaplasia and dysplasia among AEC. This can
be one of the reasons of high p-Akt activity in exfoliated
epithelial cells in biomass users, because p-Akt expression
has been found markedly increased in metaplasia and dys-
plasia of AEC, particularly the latter (Tsao et al. 2003). Ele-
vated p-Akt activity has been demonstrated in metaplastic
and dysplastic areas of the bronchial epithelium of patients
with lung cancer, whereas normal and hyperplastic bron-
chial epithelia of these patients exhibited little or no activity
(Balsara et al. 2004). Compared with metaplastic and dys-
plastic cells that were 44–88%positive for p-Akt, Tsao
et al. (2003) found that only 33%of non-small cell lung
cancer specimens expressed p-Akt. It was hypothesized that
Akt activation is an early and frequent event in lung cancer
development (Tsao et al. 2003; Balsara et al. 2004). In sup-
port of this, pharmacological inhibition of PI3K and conse-
quent down-regulation of Akt phosphorylation has been
shown to inhibit premalignant and malignant growth in
human bronchial epithelium (Chun et al. 2003). In view
of these reports, overexpression of phosphorylated (active)
Akt in exfoliated airway cells of biomass users indicate
greater risk of bronchogenic carcinoma in these women.
Indeed, chronic exposures to biomass smoke have been
implicated as a major risk factor for lung cancer among
women in India (Smith 2000; Smith and Mehta 2003;
Behera and Balamugesh 2005). Currently, lung cancer is the
fifth leading site of cancer among nonsmoking women in
eastern India, where this study was conducted (Nandakumar
et al. 2004). Since millions of rural women of the country
are exposed to IAP from biomass smoke, the present find-
ings have public health relevance and warrant immediate
measures to reduce IAP from BMF use.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support
received from Central Pollution Control Board, Government
of India, Delhi, in carrying out this study.
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