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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

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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 significantly 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 reactive oxygen species and concomitant depletion of superoxide dismutase. Measurement of particulate matter having a diameter of less than 10 and 2.5 µm 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.
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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
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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
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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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... 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]. ...
... Activation of the pro-growth PI3K-Akt signaling pathway could be a significant factor in this regard, since Akt activation contributes to epithelial dysplasia [219], lung cancer [220,221] and metastasis [219]. Considering these, it has been suggested that p-Akt [215] and proinflammatory cytokines [239] could be used as potential biomarkers of IAPassociated lung cancer among nonsmokers. ...
Chapter
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.
... 10 Biomass smoke is associated with markedly elevated particulate matter (PM) exposure 14 which may increase the risk of atherosclerosis and CVD via similar mechanisms to outdoor air pollutionnamely, lung-mediated inflammation, autonomic dysfunction and oxidative stress. [15][16][17] Long-term prospective cohort studies of outdoor air pollution have shown an association between elevated PM concentrations and an increased risk of atherosclerosis and death from CVD. [18][19][20][21] Population-based studies have found that CVD is strongly associated with increased carotid artery intima-media thickness (CIMT), which is a robust intermediate phenotype of atherosclerosis, [22][23][24][25][26][27] and carotid atherosclerotic plaques. We therefore used carotid artery ultrasound to test our primary hypothesis that biomass fuel smoke exposure is associated with increased CIMT and increased prevalence of carotid artery atherosclerotic plaques, and measured blood pressure to evaluate our secondary hypothesis that biomass fuel exposure is associated with higher blood pressure. ...
... 10 Principally, the mechanisms involve increases in oxidative stress, lung-mediated inflammation and stimulation of the autonomic nervous system. 40 Despite a paucity of controlled studies in the field of biomass fuel smoke exposure in humans, recent studies have demonstrated that exposure is associated with increased airway inflammation, 16 serum amyloid A, 15 inflammatory cytokines, circulating neutrophils, 17 oxidised LDL particles and reactive oxygen species, 11 all of which are associated with the development of atherosclerosis. Intriguingly, however, the biomass fuel group in our study actually demonstrated less inflammation (lower hsCRP) than those in the clean fuel group. ...
Article
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BACKGROUND: Biomass fuels are used for cooking in the majority of rural households worldwide. While their use is associated with an increased risk of lung diseases and all-cause mortality, the effects on cardiovascular disease (CVD) are not well characterised. Exposure to biomass fuel smoke has been associated with lung-mediated inflammation and oxidative stress, which may increase the risk of atherosclerosis as evaluated by carotid intima-media thickness (CIMT), carotid atherosclerotic plaque prevalence and blood pressure. METHODS: A cross-sectional study was performed in 266 adults aged ≥35 years in Puno, Peru (3825 m above sea level). We stratified participants by their long-term history of exposure to clean fuel (n=112) or biomass fuel (n=154) and measured 24 h indoor particulate matter (PM2.5) in a random subset (n=84). Participants completed questionnaires and underwent a clinical assessment, laboratory analyses and carotid artery ultrasound. The main outcome measures were CIMT, carotid plaque and blood pressure. RESULTS: The groups were similar in age and gender. The biomass fuel group had greater unadjusted mean CIMT (0.66 vs 0.60 mm; p<0.001), carotid plaque prevalence (26% vs 14%; p=0.03), systolic blood pressure (118 vs 111 mm Hg; p<0.001) and median household PM2.5 (280 vs 14 µg/m(3); p<0.001). In multivariable regression, the biomass fuel group had greater mean CIMT (mean difference=0.03 mm, 95% CI 0.01 to 0.06; p=0.02), a higher prevalence of carotid plaques (OR=2.6, 95% CI 1.1 to 6.0; p=0.03) and higher systolic blood pressure (mean difference=9.2 mm Hg, 95% CI 5.4 to 13.0; p<0.001). CONCLUSIONS: Chronic exposure to biomass fuel was associated with increased CIMT, increased prevalence of atherosclerotic plaques and higher blood pressure. These findings identify biomass fuel use as a risk factor for CVD, which may have important global health implications.
... Finally, we also measured plasma levels of oxidized low density lipoproteins (oxLDL: another indicator of oxidative stress) using ELISA (Mercodia Inc., Winston Salem, NC) to confirm that the oxidative stress was a true observable fact in the present study. All the biomarkers and techniques used to measure oxidative stress are well established by us or others and details can be found in the literatures [20,25,26,30,[32][33][34][35]. ...
Article
Introduction: Oxidative stress and platelet integrin α2bβ3 plays important role in the process of hemostasis and thrombosis. We hypothesized that device-induced patient specific oxidative stress and integrin α2bβ3 shedding may be linked to major bleeding complication (MBC) in heart failure (HF) patients supported by continuous flow left ventricular assist devices (CF-LVADs). Materials and methods: We recruited 47patients implanted with CF-LVADs and 15 healthy volunteers. Fourteen patients developed MBC (bleeder group) within one month after implantation while others were considered non-bleeder group (n=33). Oxidative stresses were evaluated by measuring reactive oxygen species (ROS) in platelets, superoxide dismutase (SOD) activity, total antioxidant capacity (TAC) and oxidized low density lipoprotein (oxLDL). Assessments of α2bβ3 were carried out using flow cytometry and ELISA. Results: Biomarkers of oxidative stress and α2bβ3 shedding (decreased surface expression and higher plasma levels) were found to be preexisting condition in all HF patients prior to CF-LVAD implantation compared to the healthy volunteers. Significantly elevated levels of ROS and oxLDL; concomitant depletion of SOD and TAC; and α2bβ3 shedding were observed in the bleeder group temporarily in comparison to the non-bleeder group after CF-LVAD implantation. A significantly strong association between α2bβ3 shedding and biomarkers of oxidative stress was observed; suggesting a potential role of oxidative stress in platelet integrin shedding leading to MBC after CF-LVAD implantation. Moreover, a receiver operating characteristic (ROC) analysis indicated that the likelihood of MBC data from Integrin α2bβ3 shedding had a predictive power of MBC in CF-LVAD patients. Conclusions: Oxidative stress might play a potential role in accelerating α2bβ3 shedding and platelet dysfunction, resulting in MBC in CF-LVAD patients. Integrin α2bβ3 shedding may be used to refine bleeding risk stratification in CF-LVAD patients.
... SOD activity was calculated from the standard curve and was expressed as U/mL RBC lysate. Details of the ROS and SOD assessment procedures can be found in our previously published literature [26][27][28][29]. ...
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Millions of poor people in the developing world still thrive on ragpicking. In the present study, we have examined whether ragpicking is associated with increased risk of cardiovascular disease. For this, we have enrolled 112 premenopausal female ragpickers (median age 30 years) and 98 age-matched housemaids as control from Kolkata, Eastern India. Venous blood was drawn for routine hematology; flow cytometry was used to measure generation of reactive oxygen species (ROS) by leukocytes, surface expression of CD62P (P-selectin) in platelets and CD11b in leukocytes. Collagen-induced platelet aggregation was evaluated by aggregometer, and erythrocytic superoxide dismutase (SOD) was measured by spectrophotometry. Soluble P-selectin (sP-sel) and CD40L (sCD40L), neutrophil-activating protein-2 (NAP-2), platelet and plasma serotonin, oxidized low-density lipoprotein (oxLDL), and anticardiolipin antibodies (aCL) in plasma were measured by ELISA. Compared with control, the ragpickers had significantly higher prevalence of hypertension and prehypertension, and hypertension was positively associated with ragpicking. The ragpickers also had higher levels of inflammation (elevated NAP-2), oxidative stress (elevated ROS generation with depleted SOD) with oxLDL, platelet activation and aggregability, soluble CD40 ligand, with altered serotonin level (rose in plasma but depleted in platelet). A greater percentage of ragpickers had elevated serum level of aCL of the IgG and IgM isotypes than the controls. The results suggest that the occupation of ragpicking increases the risk of cardiovascular diseases in premenopausal women of Eastern India via inflammation, oxidative stress, platelet hyperactivity, and hypertension.
... Results were reported as average concentrations plus/minus standard deviation (±SD). Raw DustTrak data were corrected as determined by previous studies (Mondal et al. 2010). MiniVol with PM 2.5 or PM 10 impactor heads (Airmetrics, Eugene, OR) collected air samples at 4 L/min onto 47-mm quartz filters (Pallflex Tissuquartz) for 24-hours for subsequent BC and biopolymer determinations. ...
Article
A pilot study was conducted to determine the levels of several air pollutants inside the homes in the small highlands (Andean) town of Langui, Peru. The measured pollutants included fine particulate matter (PM 2.5) and black carbon (BC) in addition to carbohydrate and protein levels, which were used as markers for biological aerosols. Sources of indoor aerosols mainly included combustion in traditional biomass stoves, indoor biomass fuel storage, and small animals. An initial assessment of health outcomes (respiratory disease) in this population was conducted through surveys in 30 homes. In addition, a modified traditional stove was evaluated and indicated a 99% reduction in PM2.5 levels over a 24hour period and a 96% reduction in BC emissions during cooking. This study demonstrated that significant improvements in indoor air quality can be achieved using local resources.
... However, the absolute burden of disease is larger in men due to larger underlying disease rates in men [94]. Evidence suggests that indoor air pollution mainly caused by burning of biomass fuel can increase oxidative stress and impair several immune functions in the respiratory tract [31,93,[98][99][100][101][102][103]. ...
Article
Our bodies are normally well protected against continual attack from pathogens and noxious insult by a complex and integrated immune system. However, daily bombardment from indoor and outdoor air pollutants can compromise immune function and ultimately lead to infection (e.g. acute respiratory tract infections, diarrhoea) and conditions such as sick building syndrome (with mucosal, skin and general symptoms). All of us may be affected by reduced air quality, although certain factors increase the risk of impaired immunity (e.g. young or advancing age, exposure to tobacco smoke, close proximity to areas of high air pollution, office work, commuting). A major exogenous factor modulating immune function is nutrition; even subclinical deficiencies in various nutrients can have adverse effects on the immune system, which may be exacerbated by environmental threats. In particular, the oxidant-antioxidant balance (vital for communication within the immune system) may be affected. Dietary supplementation can help to restore immune function to the normal range, and an antioxidant-containing multivitamin supplement has been shown to ameliorate the symptoms of sick building syndrome, acute respiratory tract infections and diarrhoea. This review looks at the impact of reduced air quality on the oxidant-antioxidant balance and the role of selected micronutrients (vitamins A, D, E, C, B6, B12, folate and the trace elements copper, iron, selenium and zinc) and multivitamin supplementation.
... Wood-smoke particles produced more extensive DNA damage as measured by strand breaks and formamidopyrimidine DNA glycosyl ase sites than traffic-generated particulate matter in a laboratory-based study conducted on lung epithelium and monocytic cell lines (Danielsen et al. 2009). An analysis of samples collected from participants in a case-control study of Indian women (n = 172) suggested that chronic exposure to biomass smoke activates Akt, a protein that has been implicated in the development of a wide range of human cancers (Mondal et al. 2010). ...
Article
Full-text available
Background: Indoor air pollution (IAP) derived largely from the use of solid fuels for cooking and heating affects about 3 billion people worldwide, resulting in substantial adverse health outcomes, including cancer. Women and children from developing countries are the most exposed populations. A workshop was held in Arlington, Virginia, 9–11 May 2011, to better understand women’s and children’s potential health effects from IAP in developing countries. Workshop participants included international scientists, manufacturers, policy and regulatory officials, community leaders, and advocates who held extensive discussions to help identify future research needs. Objectives: Our objective was to identify research opportunities regarding IAP and cancer, including research questions that could be incorporated into studies of interventions to reduce IAP exposure. In this commentary, we describe the state of the science in understanding IAP and its associations with cancer and suggest research opportunities for improving our understanding of the issues. Discussion: Opportunities for research on IAP and cancer include studies of the effect of IAP on cancers other than lung cancer; studies of genetic factors that modify susceptibility; studies to determine whether the effects of IAP are mediated via germline, somatic, and/or epigenetic changes; and studies of the effects of IAP exposure via dermal and/or oral routes. Conclusions: IAP from indoor coal use increases the risk of lung cancer. Installing chimneys can reduce risk, and some genotypes, including GSTM1-null, can increase risk. Additional research is needed regarding the effects of IAP on other cancers and the effects of different types of solid fuels, oral and dermal routes of IAP exposure, genetic and epigenetic mechanisms, and genetic susceptibility.
Article
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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.
Article
We evaluated AgNOR expression in airway epithelial cells (AECs) as a risk factor of lung carcinogenesis in 228 nonsmoking women exposed to biomass fuel (BMF). A total of 185 age-matched women who cooked with cleaner fuel (liquefied petroleum gas [LPG]) were enrolled as study controls. Compared with controls, Papanicolaou-stained sputum samples showed 4 and 8 times higher prevalence of metaplasia and dysplasia, respectively, in AECs of BMF users. AgNOR staining showed significantly larger numbers of dots and larger size and percentage of AgNOR-occupied nuclear area in normal AECs of BMF users than in controls. Interestingly, AgNOR parameters increased dramatically when the cells were transformed from normalcy to metaplasia and dysplasia. Compared with LPG users, BMF users showed a marked rise in reactive oxygen species (ROS) generation and a depletion of superoxide dismutase (SOD), indicating oxidative stress. Indoor air of BMF-using households had 2-5 times more particulate pollutants (PM10 and PM2.5), 73% more nitrogen dioxide (NO2), and 4 times more particulate-laden benzo(a)pyrene [B(a)P], but no difference in sulfur dioxide was observed. A high-performance liquid chromatography (HPLC) study estimated a 6-fold rise in benzene metabolite trans, trans-muconic acid (t,t-MA) in urine of BMF users. After controlling confounding factors using multivariate logistic regression, positive associations were observed between cellular changes, AgNOR parameters, and PM10, PM2.5, NO2, B(a)P, and t,t-MA levels, especially the concentration of B(a)P. In conclusion, cumulative exposure to biomass smoke causes oxidative stress and enhances AgNOR expression in precancerous metaplastic and dysplastic AECs and appears to be a risk factor for developing lung cancer.
Article
This is a cross-sectional review of biomarkers used in air pollution research from January 2009 through December 2012. After an initial keyword search in PubMed retrieving 426 articles, a comprehensive abstract review identified 54 articles of experimental design that used biomarkers of exposure or effect in human studies in the area of air pollution research during this specified time period. A thorough bibliographic search of the included articles retrieved an additional 65 articles meeting the inclusion criteria. This review presents these 119 studies and the 234 biomarkers employed in these air pollution research investigations. Data presented are 70 biomarkers of exposure with 54% relating to polycyclic aromatic hydrocarbons, 36% volatile organic carbons, and 10% classified as other. Of the 164 biomarkers of effect, 91 and 130 were used in investigating effects of short-term and chronic exposure, respectively. Results of biomarkers used in short-term exposure describe different lag times and pollutant components such as primary and secondary pollutants, and particle number associated with corresponding physiological mechanisms including airway inflammation, neuroinflammation, ocular, metabolic, early endothelial dysfunction, coagulation, atherosclerosis, autonomic nervous system, oxidative stress, and DNA damage. The review presents three different exposure scenarios of chronic, occupational, and extreme exposure scenarios (indoor cooking) with associated biomarker findings presented in three broad categories of (1) immune profile, (2) oxidative stress, and (3) DNA damage. This review offers a representation of the scope of data being explored by air pollution researchers through the use of biomarkers and has deliberately been restricted to this particular subject rather than an extensive or in-depth review. This article provides a contextualization of air pollution studies conducted with biomarkers in human subjects in given areas while also integrating this complex body of information to offer a useful review for investigators in this field of study.
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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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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.
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The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27KIP1 expression have been detected in most advanced prostate cancers1, 2. But mice deficient for Cdkn1b (encoding p27Kip1) do not develop prostate cancer3, 4, 5. PTEN activity leads to the induction of p27KIP1 expression, which in turn can negatively regulate the transition through the cell cycle6. Thus, the inactivation of p27KIP1 may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten +/-/Cdkn1b -/- mice. Moreover, Pten +/-/Cdkn1b -/- mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27Kip1 through the control of cell-cycle progression.
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