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Neutrophilic Inflammatory Response and Oxidative Stress in Premenopausal Women Chronically Exposed to Indoor Air Pollution from Biomass Burning


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The possibility of inflammation and neutrophil activation in response to indoor air pollution (IAP) from biomass fuel use has been investigated. For this, 142 premenopausal, never-smoking women (median age, 34 years) who cook exclusively with biomass (wood, dung, crop wastes) and 126 age-matched control women who cook with cleaner fuel liquefied petroleum gas (LPG) were enrolled. The neutrophil count in blood and sputum was significantly higher (p < 0.05) in biomass users than the control group. Flow cytometric analysis revealed marked increase in the surface expression of CD35 (complement receptor-1), CD16 (F(C)γ receptor III), and β(2) Mac-1 integrin (CD11b/CD18) on circulating neutrophils of biomass users. Besides, enzyme-linked immunosorbent assay showed that they had 72%, 67%, and 54% higher plasma levels of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-6, and interleukin-12, respectively, and doubled neutrophil chemoattractant interleukin-8. Immunocytochemical study revealed significantly higher percentage of airway neutrophils expressing inducible nitric oxide synthase, while the serum level of nitric oxide was doubled in women who cooked with biomass. Spectrophotometric analysis documented higher myeloperoxidase activity in circulating neutrophils of biomass users, suggesting neutrophil activation. Flow cytometry showed excess generation of reactive oxygen species (ROS) by leukocytes of biomass-using women, whereas their erythrocytes contained a depleted level of antioxidant enzyme superoxide dismutase (SOD). Indoor air of biomass-using households had two to four times more particulate matter with diameters of <10 μm (PM(10)) and <2.5 μm (PM(2.5)) as measured by real-time laser photometer. After controlling potential confounders, rise in proinflammatory mediators among biomass users were positively associated with PM(10) and PM(2.5) in indoor air, suggesting a close relationship between IAP and neutrophil activation. Besides, the levels of neutrophil activation and inflammation markers were positively associated with generation of ROS and negatively with SOD, indicating a role of oxidative stress in mediating neutrophilic inflammatory response following chronic inhalation of biomass smoke.
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1 23
ISSN 0360-3997
Volume 35
Number 2
Inflammation (2012) 35:671-683
DOI 10.1007/s10753-011-9360-2
Neutrophilic Inflammatory Response
and Oxidative Stress in Premenopausal
Women Chronically Exposed to Indoor Air
Pollution from Biomass Burning
Anirban Banerjee, Nandan Kumar
Mondal, Debangshu Das & Manas
Ranjan Ray
1 23
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Neutrophilic Inammatory Response and Oxidative Stress
in Premenopausal Women Chronically Exposed to Indoor
Air Pollution from Biomass Burning
Anirban Banerjee,
Nandan Kumar Mondal,
Debangshu Das,
and Manas Ranjan Ray
AbstactThe possibility of inammation and neutrophil activation in response to indoor air poll-
ution (IAP) from biomass fuel use has been investigated. For this, 142 premenopausal, never-
smoking women (median age, 34 years) who cook exclusively with biomass (wood, dung, crop
wastes) and 126 age-matched control women who cook with cleaner fuel liqueed petroleum gas
(LPG) were enrolled. The neutrophil count in blood and sputum was signicantly higher (p< 0.05)
in biomass users than the control group. Flow cytometric analysis revealed marked increase in the
surface expression of CD35 (complement receptor-1), CD16 (F
γreceptor III), and β
integrin (CD11b/CD18) on circulating neutrophils of biomass users. Besides, enzyme-linked imm-
unosorbent assay showed that they had 72%, 67%, and 54% higher plasma levels of the proina-
mmatory cytokines tumor necrosis factor-alpha, interleukin-6, and interleukin-12, respectively, and
doubled neutrophil chemoattractant interleukin-8. Immunocytochemical study revealed signicantly
higher percentage of airway neutrophils expressing inducible nitric oxide synthase, while the serum
level of nitric oxide was doubled in women who cooked with biomass. Spectrophotometric analysis
documented higher myeloperoxidase activity in circulating neutrophils of biomass users, suggesting
neutrophil activation. Flow cytometry showed excess generation of reactive oxygen species (ROS)
by leukocytes of biomass-using women, whereas their erythrocytes contained a depleted level of
antioxidant enzyme superoxide dismutase (SOD). Indoor air of biomass-using households had two
to four times more particulate matter with diameters of <10 μm (PM
) and <2.5 μm (PM
measured by real-time laser photometer. After controlling potential confounders, rise in proin-
ammatory mediators among biomass users were positively associated with PM
and PM
indoor air, suggesting a close relationship between IAP and neutrophil activation. Besides, the levels
of neutrophil activation and inammation markers were positively associated with generation of
ROS and negatively with SOD, indicating a role of oxidative stress in mediating neutrophilic inf-
lammatory response following chronic inhalation of biomass smoke.
KEY WORDS: biomass fuel; neutrophil; inammation; oxidative stress; premenopausal women; India.
A large majority of rural women of the developing
world including India are chronically exposed to high
levels of indoor air pollution (IAP) from daily household
cooking with traditional biomass fuel (BMF) such as
dried cow dung cake, rewood, and agricultural wastes.
Smoke emitted from burning biomass contains a wide
ABBREVIATIONS: ACD, acid citrate dextrose; BMF, biomass fuel;
BSA, bovine serum albumin; DCF-DA, dichlorouorescein diacetate;
EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immu-
nosorbent assay; FACS, uorescence-activated cell sorter; FITC,
uorescein isothiocyanate; HRP, horseradish peroxidase; IAP, indoor
air pollution; ICC, immunocytochemistry; IL, interleukin; iNOS,
inducible nitric oxide synthase; LPG, liqueed petroleum gas; MFI,
mean uorescence intensity; MPO, myeloperoxidase; NO, nitric oxide;
PAP, Papanicolaou; PBS, phosphate-buffered saline; PE, phycoerythrin;
PM, particulate matter; ROS, reactive oxygen species; SOD, super-
oxide dismutase; TNF, tumor necrosis factor
Anirban Banerjee and Nandan Kumar Mondal contributed equally to
this work.
Department of Experimental Hematology, Chittaranjan National
Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700 026, India
To whom correspondence should be addressed at Department of
Experimental Hematology, Chittaranjan National Cancer Institute, 37,
S. P. Mukherjee Road, Kolkata, 700 026, India. E-mail: nandan_
0360-3997/12/0200-0671/0 #2011 Springer Science+Business Media, LLC
Inammation, Vol. 35, No. 2, April 2012 (#2011)
DOI: 10.1007/s10753-011-9360-2
Author's personal copy
spectrum of pollutants that include carbon monoxide,
coarse, ne, and ultrane particulate matters (PM),
oxides of nitrogen and sulfur, formaldehyde, transitional
metals, and volatile organic compounds including
benzene and polycyclic aromatic hydrocarbons such as
benzo(a)pyrene [1]. Some of these pollutants like benzo
(a)pyrene and benzene are potent human carcinogens
[2]. Women who used to cook with BMF in poorly
ventilated kitchen for 26 h/day are believed to be
inhaling carcinogens equivalent to smoking two packs of
cigarettes per day [3]. Epidemiological studies have
shown that exposure to ne particles promotes inam-
mation in the lung [49] via activation of alveolar
macrophages and lung epithelial cells [10,11]. Such
exposures may also elicit systemic inammatory
response [12], associated with increased levels of several
cytokines (interleukin-6 and -1b) in the bloodstream [13]
as well as increased production, release, and activation
of neutrophils from the bone marrow [14].
Neutrophils are the most abundant leukocytes
present in human blood. These cells are important for
phagocytosis and destruction of invading pathogens,
especially bacteria [15]. They are among the rst cells to
leave the bloodstream and reach the tissues in case of
tissue injury [16]. During the beginning of an acute
phase of inammation, particularly as a result of
bacterial infection, environmental exposure, and some
cancers, neutrophils are among the rst responders that
migrate towards the site of inammation. Such migration
of blood leukocytes to the tissues in order to ght
against invading pathogens and to mediate tissue repair
is known as inammation. Neutrophils have been
implicated in the development of bronchial hyperres-
ponsiveness [17]. Neutrophilic inammation in the
airways and the bronchial submucosa has been reported
in patients with severe asthma [18,19], especially during
exacerbation [20]. Neutrophil elastase has the capacity to
induce mucus gland hyperplasia and mucus secretion
[21] and proliferation or apoptosis of airway smooth
muscle cells [22,23]. Myeloperoxidase (MPO) is an
enzyme found in myeloid cells, particularly in neutro-
phils, and to a lesser extent in monocytes and tissue
macrophages. It plays an important role in the host
defense against bacteria and viruses. In neutrophils,
MPO is located within the primary granules and its
concentration is used as an indicator of neutrophil
activation and corresponding inammatory response
[24]. Neutrophil migration from circulation into an area
of inammation involves regulated expression of a
number of adhesion molecules on the cell surface.
Upregulation of these surface molecules has been
reported in patients with chronic obstructive pulmonary
disease (COPD) [25]. A recent study has shown allergic
pulmonary inammation following exposure to diesel
exhaust particles [26]. Moreover, studies in laboratory
animals have documented lung inammation induced by
air pollution [27]. In view of these reports, it is
reasonable to assume that IAP from biomass burning
may lead to the activation of circulating neutrophils and
consequent inammatory response in women who cook
with these fuels.
Neutrophils in peripheral blood may be activated or
primed with regard to superoxide production, chemotaxis,
and increased expression of surface molecules such as
CD35 which mediates the binding and phagocytosis of
C3b-coated particles and immune complexes [28]. The
study of Smith et al.[29] suggested that upregulation of
CD11b expression may be associated with neutrophils
degranulation. Data from studies of pathological condi-
tions suggest that neutrophil receptor expression is altered
in response to inammatory stimuli and tissue injury. For
example, the expression of CD11b and CD35 receptors is
enhanced in patients suffering sepsis. In contrast, the
expression of CD11b, CD35, and CD16 is reduced in
patients who have experienced acute trauma and burns.
These alterations may represent adaptive responses to
control the level of neutrophils and complement activation
during these pathological conditions [3032]. Alterations
in neutrophil receptor expression could also affect neu-
trophil functional activity. For example, a reduction in the
neutrophil expression of CD11b and CD16 in patients with
thermal injuries has been associated with defects in
respiratory burst activity, and this has resulted in an
increased susceptibility to infection [33]. Neutrophils
respond to infection and tissue injury by recognizing and
binding immunoglobulin G (IgG) molecules and comple-
ment proteins that coat the surface of foreign pathogens
and host tissue fragments. This recognition-binding proc-
ess is mediated by neutrophil surface receptors, including
the complement receptor 1 CD35, the complement
receptor 3 CD11b, and the low-afnity IgG receptor
CD16. The engagement of these surface receptors initiates
a cascade of intracellular events leading to the release of
enzymes (degranulation) and reactive oxygen species
(ROS; respiratory burst activity) from neutrophils [34].
Together, these enzymes and ROS assist in the destruction
and degradation of foreign pathogens and damaged tissue
fragments [35].
Our earlier studies have linked IAP from BMF with
upregulation of ribosome biogenesis [36,37], inappro-
672 Banerjee, Mondal, Das, and Ray
Author's personal copy
priate response of DNA damage repair proteins in the
face of increased DNA damage [38,39], overactivation
of signal transduction pathway involving Akt, i.e.,
protein kinase B [40], and hypertension with elevated
levels of oxidized low-density lipoprotein indicating
cardiovascular disease [41]. These ndings along with
observations from other studies led us to hypothesize
that chronic exposure to IAP from biomass burning
could be associated with neutrophilic inammatory
responses and oxidative stress in premenopausal women
of rural India who cook regularly with BMF. To our
knowledge, no such study has been carried out in India
or any other developing country where biomass is
extensively used as a major source of household energy.
Against this background, we examined in this study the
impact of IAP from BMF use on neutrophil activation 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
ndings with an age-matched group of control women
from same neighborhood who cooked with cleaner fuel
liqueed petroleum gas (LPG).
A total of 268 premenopausal women aged between
23 and 41 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 organized in different villages
with the active cooperation of the local administrative
bodies and nongovernmental organizations. Among the
participants, 142 women (age, 2340 years; median age,
34 years) were cooking daily for 2.55.5 h exclusively
with wood, cow dung, and agricultural refuse, such as
bamboo, jute stick, paddy husk, hay, and dried leaves for
the past 5 years or more. Accordingly, they were
grouped as biomass users. The remaining 126 women
(age, 2441 years; median age, 33 years) were from the
same villages but they used to cook with cleaner fuel
LPG and were considered as control.
Inclusion and Exclusion Criteria
The inclusion criteria 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 (BMI) >15 and <30 kg/m
. 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 information 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 problems in the past 3 months
and last 1 year. As most of the participants 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.
Collection of Blood and Expectorated Sputum
Venous blood (5 ml) was collected in vacutainer tubes
(Becton Dickinson [BD], USA) containing K
anticoagulant. Blood was collected at a xed time of the
day (09:30 AM 10:30 AM) to minimize diurnal variation.
A drop of blood was used for smear preparation on clean
glass slide for each individual. An aliquot of whole blood
was mixed with acid citrate dextrose (ACD) solution in the
ratio of 5:1 separately in sterile vacutainer tubes for
isolation of circulation neutrophils. Blood in the no-
additive vacutainer was allowed to clot and serum was
collected. Plasma was obtained by centrifugation at
2,500×gfor 10 min at 4°C.
Early morning expectorated sputa were collected in
sterile plastic cups for three consecutive days to harvest
airway cells following the procedure of Erkilic et al.
[42]. Two smears were prepared from the nontransparent
highly viscous part of each freshly collected sputum
sample on clean glass slides from each days sample
one for Papanicolaou (PAP) staining and the other for
immunocytochemistry (ICC) of inducible nitric oxide
synthase (iNOS). After air drying, one slide marked for
PAP staining was xed with 95% ethanol and the other
for ICC were xed in ice-cold methanol for 20 min at
the site of collection. The remaining part of the
expectorated sputum was collected in sterile plastic
screw-cap tubes containing 20 ml of phosphate-buffered
saline (PBS) with 0.1% dithiotheritol.
Routine Hematology
Differential counts of leukocytes were done from
Leishman-stained blood lms. Routine hematological
parameters, such as total and differential counts of white
blood cells and total count of platelets, were carried out
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using a hemocytometer under light microscope (Leitz,
PAP Staining for Routine Sputum Cytopathology
Ethanol-xed slides were brought to the laboratory
and were stained with PAP following the procedure of
Hughes and Dodds [43]. The slides were coded and
examined under light microscope (Leitz, Germany) at
×400 and ×1,000 magnications. At least 10 high-power
elds (hpf; ×40 objective and ×10 eyepiece) per slide
were examined. Sputum cytology and differential dis-
tribution of inammatory cells were performed follow-
ing the established criteria [44].
Isolation of Circulating Neutrophils
Neutrophils were isolated following the procedure
of Drabikova et al.[45] with slight modications. In
brief, blood in ACD was added with 6% dextran
solution in 0.9% NaCl (blooddextran ratio, 1:2 v/v).
Then, it was allowed to sediment for 45 min at room
temperature. The supernatant was centrifuged at 700×g
for 12 min at 4°C, the cell pellet was reconstituted in
1.2 ml ice-cold distilled water, and within 20 s, 400 μlof
0.6 M KCl was added and mixed. It was diluted to 5 ml
with PBS and kept in ice for 5 min for red blood cell
(RBC) lysis. It was then centrifuged at 770×g for 6 min
at 4°C. Supernatant was discarded and pellet resus-
pended in 12 ml of ice-cold PBS. Next, the cell
suspension was layered over 3 ml of FicollHistopaque
(Sigma Aldrich, USA) in a 15-ml tube and centrifuged at
1,200×g for 30 min. After discarding the supernatant,
the pellet was dissolved in 500 μl of ice-cold Hanks
balanced salt solution. About 99% of neutrophils were
isolated by this procedure and viability of the cells was
>90% in trypan blue dye exclusion test.
Assessment of the Expression of Surface Molecules
by Flow Cytometry
Whole blood (25 μl) was added to polypropylene
tubes and incubated for 20 min at room temperature in
the dark with FITC-conjugated antihuman CD11b,
CD18, and CD35 monoclonal antibodies, PE-conjugated
antihuman CD16 monoclonal antibody (BD, USA), and
isotype controls. Thereafter, the erythrocytes were lysed
with 2 ml of 1X FACS lysing solution (BD, USA), and
samples were centrifuged at 500×gfor 5 min. The cell
pellet was washed with ice-cold PBS containing 0.1%
sodium azide and resuspended in 500 μlof1%
paraformaldehyde in PBS. The samples were analyzed
in a ow cytometer (FACSCalibur with Sorter, BD, San
Jose, CA, USA) using Cell Quest software (BD, USA).
A gate was drawn around the granulocytes showing
neutrophils as CD16-positive cells with eosinophils as
CD16-negative cells with high side scatter. A further
gate was then drawn around the neutrophil (CD16+) or
eosinophil (CD16) populations and reected into a
histogram so that cells could be labeled with a further
uorochrome (e.g., CD11b or CD18). Fluorescence was
measured with a FACSCalibur ow cytometer and Cell
Quest software (BD, USA). Measurements were made
on the FL1 (green) and FL2 (red) channels, and the gates
were adjusted to the negative control quadrant. A total of
15,000 events were collected. Results were recorded as
mean uorescence intensity (MFI), which represents the
cell surface receptor density.
Measurement of Proinammatory Cytokine
and Chemokine by ELISA
The levels of proinammatory cytokine tumor
necrosis factor-alpha (TNF-α), interleukin-6 (IL-6),
interleukin-12 (IL-12), and chemokine interleukin-8
(IL-8) were measured in blood plasma and nitric oxide
(NO) level in serum by ELISA using commercially
available kits of BD Biosciences Pharmingen, San
Diego, USA (for TNF-α, IL-6, and IL-12), Roche
Diagnostics GmbH, Germany (for IL-8), and Oxford
Biomedical Research, Oxford, MI, USA (for NO). The
assays were performed following the procedures recom-
mended by the manufacturers. The instructions of the
manufacturers were followed during the study and
readings were taken in microplate reader (Bio-Rad,
USA) at 450 nm.
Immunocytochemical Localization of iNOS
ICC of iNOS in isolated blood neutrophils and
sputum cells were done following a standard procedure
[38,40]. In brief, slides were air dried and xed in ice-
cold methanol for 30 min, air dried, washed in PBS
thrice, and blocked in 3% BSA (Sigma-Aldrich Chem-
icals, Saint Louis, MO, USA) for 1 h at room temper-
ature. The slides were overnight incubated in darkness
with rabbit polyclonal iNOS (Santa Cruz, CA, USA)
primary antibody (diluted 1:100 in 1% BSA, respec-
tively) in a humid box at 4°C. After washing with PBS,
slides were incubated with antirabbit IgG, F(ab)2-HRP
(Santacruz, USA) secondary antibody diluted 1:500 in
1% BSA for 90 min. The slides were developed by
674 Banerjee, Mondal, Das, and Ray
Author's personal copy
incubating with substrate for HRP for 45 min in
darkness followed by washing with distilled water and
counterstaining with hematoxylin, dehydration in graded
ethanol, and mounted in distrene plasticizer xylene. The
slides were coded and examined blindly. For each
participant, one blood neutrophil sample and three sputa
samples obtained from three consecutive days were
examined. The 3-day average values of iNOS expres-
sions in sputum cells were taken as the representative
data of each participant.
Quantitative Measurement of MPO Activity
by Spectrophotometry
MPO activity in isolated blood neutrophil homoge-
nate was determined by a modication of the assay
method involving O-dianisidine [46]. The principle
involves oxidation of O-dianisidine by MPO present in
the isolated neutrophil homogenate to produce a color
complex which is read spectrophotometrically at
460 nm. The higher the MPO activity, the more oxygen
released from hydrogen peroxide; hence, more oxidation
of O-dianisidine and consequently higher observed
spectrophotometer reading at 460 nm. The assay mixture
contained 0.3 ml of 0.1 M phosphate buffer (pH 6.0),
0.3 ml of 0.01 M H
, 0.5 ml 0.02 M of O-dianisidine
(Sigma Chem, USA; freshly prepared in deionized
water), and 10 μl neutrophil homogenate in a nal
volume of 3 ml. The neutrophil homogenate was added
last in a cuvette with a path length of 1 cm and the
change in absorbance in 460 nm was followed for
10 min. One unit of MPO is dened as that giving an
increase in absorbance of 0.001 per minute and specic
activity is given as international units per milligram of
Flow Cytometric Measurement of ROS Generation
Generation of ROS was measured in leukocytes
present in anticoagulated venous blood and sputum cells
by ow cytometry using DCF-DA (Sigma Chem, USA)
following the procedure of Rothe and Valet [47] with
slight modications [40]. For this, 10,000 events were
acquired in a ow cytometer (FACSCalibur with sorter,
Becton Dickinson [BD], San Jose, CA, USA) using Cell
Quest software (BD). Respiratory burst and generation
of ROS by cells were associated with emission of green
uorescence that was recorded in uorescence channel 1
and was expressed as MFI in arbitrary units. In case of
blood, granulocytes, monocytes, and lymphocytes were
gated on the basis of their characteristic forward and side
scatters on dot plot and the MFI of each population was
recorded, while the 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.[48]. Whole sputum collected
in PBS with 0.1% dithiotheritol were used for cell lysate
preparation for SOD assay followed by our previously
published procedure [40]. Lysates were centrifuged at
15,000×gfor 10 min at 4°C in an Eppendorf micro-
centrifuge. The supernatant was collected and used for
SOD assay. Ethylenediaminetetraacetic acid (EDTA)-
anticoagulated blood was centrifuged at 200×gat 4°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). Following centrifugation at 500×gfor 10 min, the
supernatant was collected. In spectrophotometric cuv-
ette, 800 ml of triethanolamine (TEA)diethanolamine
(DEA) buffer containing 100 mM each of TEA and
DEA (Qualigens, Mumbai, India), 40 ml of 7.5 mM
nicotinamide adenine dinucleotide reduced disodium salt
(NADH, SRL, Mumbai, India), pH 7.4, 25 ml of a
mixture (1:1, v/v) of 0.2 M EDTA disodium salt (Sigma-
Aldrich Chem, St. Louis, MO, USA) and 0.1 M
manganous chloride (SD-Fine Chem, Mumbai, India),
and 100 ml of sample (RBC or sputum cell lysate) were
added and mixed well. The absorbance (OD) was
measured at 340 nm in a spectrophotometer (Shimadzu,
Kyoto, Japan) immediately (0 min) and 1, 2, 3, 4, and
5 min after the addition of mercaptoethanol. SOD
activity was calculated from the standard curve and
was expressed as units per milliliter.
Measurement of PM
and PM
in Indoor Air
PM with aerodynamic diameter less than 10 mm
) and 2.5 mm (PM
) were measured in the
cooking areas with real-time laser photometer (Dust-
TrakAerosol Monitor, model 8520, TSI Inc., Shore-
view MN, USA) that contained 10-mm nylon Dor-
Oliver cyclone and operated at a ow rate of 1.7 L/min,
measuring particle load in the concentration range of 1
to 100 mg/m
. The monitor was calibrated to the
standard ISO 12103-1 A1 test dust. We used two
monitors for simultaneous measurement of PM
. Air sampling was done in each household for
three consecutive days, 8 h/day (07:00 AM 03:00 PM)
675Neutrophilic Inammatory Response and Oxidative Stress
Author's personal copy
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 23 ft away from the open chullah (oven).
Accordingly, the monitor was placed in the breathing
zone of the cook 2.5 ft above the oor level on a wooden
stool 3 ft away from the chullah. LPG users, on the other
hand, cook in a standing position, and the monitor was,
therefore, placed at a height of 4.5 ft. Since laser
photometers make an overestimation of PM levels by
twofold to threefold [49], the raw data require reduction
using correction factors. We reduced the 8-h raw data by
dividing with a correction factor of 2.5 for PM
and 2.77 for PM
Statistical Analysis of Data
Results are presented as mean ±standard deviation
(SD) or median with range. Analysis between groups
was performed using Studentsttest, chi-square test, or
MannWhitney Utest, as applicable. The possibility of
an association between measured parameters with age,
BMI, family income, education, cooking hours per day,
lifetime duration of cooking (cooking years), kitchen
location, family size, number of smokers in the family,
use of mosquito repellant, and PM
and PM
levels in
cooking areas was tested using univariate regression
analysis. Variables that showed signicant 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 Scien-
ces for Windows, release 10.0, SPSS Inc., Chicago,
USA) and p<0.05 was considered signicant.
Demographic and socioeconomic characteristics of
the study population are summarized in Table 1. The
LPG and BMF users were comparable with respect to
age, BMI, cooking years, hours of cooking per day,
family income, environmental tobacco smoke for the
presence of a smoker in the family, food habit, and use
of mosquito repellant. However, BMF users were less
educated (p<0.05 in MannWhitney Utest) and their
family income was signicantly lower than that of their
neighbors who used to cook with LPG (p<0.05 in
Studentsttest). Moreover, 40.7% of BMF-using house-
holds did not possess a separate kitchen and they used to
cook in an area adjacent to the living room. In contrast,
only 15.3% of LPG-using households lacked a separate
kitchen (p<0.05 in chi-square test).
Particulate Pollutants in Indoor Air
The mean concentration of PM
in biomass-using
kitchen during cooking hours was 625±150 μg/m
4791,556 μg/m
97254 μg/m
) in LPG-using kitchen. The corresponding
level was 312±85 μg/m
(range, 262715 μg/m
and 77± 29 μg/m
(range, 46117 μg/m
). During
noncooking hours, PM
and PM
concentrations were
204± 98 μg/m
(range, 142311 μg/m
) and 82± 21 μg/m
(range, 66126 μg/m
), respectively, in biomass-using
(range, 45145 μg/
) and 45± 22 μg/m
(range, 2472 μg/m
) in LPG-using
kitchen. Thus, the particulate pollution levels of cooking
areas in biomass-using households were signicantly
higher than those in LPG-using households during
cooking and noncooking times (p<0.001).
Hematology and Sputum Cytology
The total leukocyte count in peripheral blood was
signicantly higher (p<0.05 in Studentsttest) in
BMF users compared with LPG-using controls (8.7±
0.72 vs. 5.2±0.83 × 10
/μl, respectively). Moreover,
BMF users had elevated number of total cells in
sputum (88.9±23.7 vs. 65.2 ±13.1 cells/hpf, p<0.05)
than controls. Blood and sputa of BMF users
contained more inammatory cells like neutrophils
(blood, 5,867± 445 vs. 3,708 ±281 cells/μl, p<0.01;
sputum, 58.9±17.4 vs. 42.3 ±10.6 cells/hpf, p<0.01),
eosinophils (blood, 552± 63 vs. 300 ±48 cells/μl, p<
0.01; sputum, 1.7± 0.7 vs. 0.4 ±0.2 cells/hpf, p<0.01),
and lymphocytes (blood, 2,100± 244 vs. 1,929±207
cells/μl, p>0.05; sputum, 4.6± 1.3 vs. 3.3±1.0 vs.
cells/hpf, p<0.05).
Higher Surface Expression of CD16, CD35,
and CD11b/CD18 in Circulating Neutrophils
of Biomass Users
Flow cytometric analysis revealed signicantly
higher MFI of CD16, CD35, CD11b, and CD18 in
circulating neutrophils of BMF users when compared
with that of LPG-using controls (p< 0.0001; Fig. 1). In
biomass users, the MFI of neutrophilic surface receptor
CD16 and CD35 was increased by 2.5-fold (3,196.1 ±
676 Banerjee, Mondal, Das, and Ray
Author's personal copy
337.5 vs. 1,282.1± 613.3 in controls) and 1.4-fold
(81.9±9.5 vs. 56.7 ± 4.2), respectively. Moreover, the
MFI of CD11b and CD18 on neutrophils was increased
by 37% and 66%, from 736.3±278.4 and 217.9± 93.1 in
controls to 1,008.2±234.3 and 362.1± 79.7 in biomass
users, respectively (Fig. 1).
Rise in Proinammatory Cytokines and Chemokine
in Biomass Users
Tab le 2shows signicantly elevated levels of
proinammatory cytokines TNF-α, IL-6, and IL-12
and chemokine IL-8 in plasma of BMF users in
comparison with that of LPG users. Compared with
LPG-using control women, biomass users had 72%,
67%, and 54% higher plasma levels of TNF-α, IL-6, and
IL-12, respectively. Moreover, the plasma level of IL-8
was doubled in biomass users (Table 2).
Overexpression of iNOS and NO in Biomass Users
iNOS enzyme was found located in the cyto-
plasm of the neutrophils. Compared with LPG users,
the percentage of iNOS-expressing isolated blood
Table 1. Demographic and Socioeconomic Characteristics of BMF- and LPG-using Women
Variable LPG users (n=126) BMF users (n=142) pvalue
Age in years, median (range) 33 (2441) 34 (2340) NS
Median BMI (kg/m
) 24.3 23.9 NS
Cooking years, median (range) 13 (525) 14 (522) NS
Cooking hours per day, median (range) 3 (2.55.5) 3 (2.55.5) NS
Years of schooling, median (range) 8 (314) 3 (010) <0.05
Homes with separate kitchen (%) 84.7 59.3 <0.05
Smoker in family (%) 43.9 44.1 NS
Use of mosquito repellant at home (%) 75.8 74.1 NS
Food habit, mixed (%) 97.2 96.9 NS
Members in family, median (range) 4 (26) 4 (27) NS
Family income per month in US dollars (mean ±SD) 58.41± 8.32 31.78±7.17 <0.05
The LPG- and biomass-using groups were compared by chi-square test (for results presented as percentages), MannWhitney Utest (for median
values with range), and Studentsttest (for mean± SD) and p<0.05 was considered signicant
nnumber of subjects, NS statistically not signicant
Fig. 1. Histograms showing the MFI of surface expressions of CD16 (a), CD35 (b), and β
Mac-1 integrin (CD11b/CD18; c,d) on circulating
neutrophils of rural women who cooked exclusively either with biomass or LPG for the past 5 years or more. Bars represent the SD of the mean;
*p<0.0001, compared with the LPG users.
677Neutrophilic Inammatory Response and Oxidative Stress
Author's personal copy
neutrophils was slightly increased in biomass users,
but the increment was not statistically signicant (5.1
±1.8% vs. 4.8± 1.3%, p>0.05inStudentsttest). In
airway neutrophils, however, iNOS expression was
increased three times over controls in women who
cooked exclusively with biomass (25.7± 8.4% vs. 8.9
±3.3% iNOS-expressing cells in sputum of control
women, p<0.0001). On the other hand, serum level
of NO was increased by twofold from 23.3±12.6 μM
in controls to 47.3±16.2 μMinbiomassusers
(Fig. 2).
Higher MPO Activity in Biomass Users
Spectrophotometric analysis revealed signicantly
higher MPO activity in blood neutrophils of biomass
users than that of LPG-using control women (0.516 ±
0.10 vs. 0.2644±0.07 IU/ml, p< 0.001).
Oxidative Stress Among Biomass Users
Flow cytometric analysis showed a signicant rise
in MFI of DCF-DA among BMF users, suggesting
increase in ROS generation in peripheral blood leuko-
cytes (neutrophils, monocytes, and lymphocytes) and
unfractionated sputum cells that contained alveolar
macrophages, epithelial cells, airway neutrophils, eosi-
nophils, and lymphocytes (Table 3). The MFI of DCF-
DA was 36% higher in peripheral blood neutrophils,
25% higher in monocytes, and 14% higher in lympho-
cytes of BMF users, relative to controls. ROS generation
was doubled in sputum cells of biomass users when
compared with that of controls (Table 3). 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<0.0001) and sputum cells
(565.3±115.2 vs. 785.4±183.8 U/ml, p< 0.0001),
respectively, in comparison with controls.
ROS, SOD, and Neutrophil Activation
Spearmans rank correlation analysis showed a
positive association between markers and mediators of
neutrophil activation and generation of ROS in biomass-
using women. Conversely, a negative association was
found between these markers and the level of antiox-
idant enzyme SOD (Table 4). However, the association
between IL-12 with ROS generation and SOD level was
not statistically signicant (p>0.05).
Particulate Pollutants and Neutrophil Activation
In univariate analysis, levels of particulate pollu-
tants (PM
and PM
) in indoor air, family size, family
income, education, and kitchen location were positively
associated with different neutrophil activation bio-
markers. After controlling confounding factors in multi-
Table 2. Comparison of Proinammatory Cytokine and Chemokine
Levels in Plasma of LPG and BMF users
Concentration in
plasma (pg/ml) LPG users (n=126) BMF users (n= 142)
Mean± SD 13.2±4.6 22.84±4.13*
Median (range) 11.8 (1.318.6) 21.1(17.929.5)**
Mean± SD 28.5±8.6 47.5± 13.2*
Median (range) 27.5 (11.239.4) 43.9 (3173.9)**
Mean± SD 118.6± 48.65 183.05± 54.6*
Median (range) 114.1 (67.1208.2) 174.8 (80.6331.0)**
Mean± SD 23.3±4.7 46.7±18.3*
Median (range) 19.6 (1.531.2) 31.5 (12.4109.5)**
*p<0.0001, compared with the LPG users in Studentsttest; **p<0.001
in MannWhitney Utest
Fig. 2. Histograms showing comparison of the levels of iNOS in neutrophils (a,b) and NO in serum (c) between LPG and BMF users. Bars represent
the SD of the mean; *p<0.0001, compared with the LPG users in Studentsttest.
678 Banerjee, Mondal, Das, and Ray
Author's personal copy
variate logistic regression, a strong positive association
was observed between neutrophil activation biomarkers
with PM
and PM
as shown in Table 5.
Correlation Between Neutrophil Activation
Biomarkers and Years of Cooking with Biomass
A strong positive correlation was found in Spear-
mans rank correlation test between years of cooking with
biomass and proinammatory cytokine TNF-α(rho [ρ]=
0.9103, p<0.0001), IL-6 (ρ=0.8251, p<0.001), and IL-12
(ρ=0.6316, p<0.001), proinammatory chemokine IL-8
(ρ=0.8797, p<0.0001), neutrophil surface receptor CD35
(ρ=0.9912, p<0.0001) and CD16 (ρ=0.9037, p<0.0001),
CD18 (ρ=0.7998, p<0.002), CD11b (ρ=0.8875, p<
0.0001), neutrophil iNOS (ρ=0.6247, p<0.012), NO (ρ=
0.5797, p<0.031), and MPO (ρ=0.9623, p<0.0001).
Moreover, there was a strong positive association
between oxidative stress and exposure years of cook-
ing with biomass because cooking years correlated
positively with generation of ROS in sputum cells, in
terms of MFI of DCF-DA (ρ=0.9738, p<0.0001), and
negatively with concentration of SOD in erythrocytes
(ρ=0.8993, p<0.0001).
The objective of this study was to examine whether
cumulative inhalation of biomass smoke activates
neutrophil towards the pathway of inammation via
oxidative stress in a group of rural premenopausal
women in India who are actively engaged in cooking
for the past 5 years of more. Signicantly increased
number of inammatory cells was found in blood and
sputa of biomass-using women than LPG users, suggest-
ing inammation. This nding tempted us to examine
whether the inammatory response was mediated via
overexpression of neutrophilic surface molecules, excess
proinammatorycytokine and chemokine release, and
other neutrophil activation biomarkers like iNOS and
MPO. As anticipated, biomass-using women of this
study had elevated circulating levels of proinammatory
cytokine TNF-α, IL-6, and IL-12 and proinammatory
chemokine IL-8. Besides, their neutrophils expressed
more CD16, CD35, and CD11b/CD18 on their surface
compared with their age- and sex-matched neighbors
who used to cook with cleaner fuel LPG. Collectively,
these ndings suggest that chronic exposures to biomass
smoke elicit inammatory response through the upregu-
lation of proinammatory mediators and neutrophil
adhesion molecules.
Table 3. Comparison of ROS Generation by Blood Leukocytes and
Sputum Cells between Biomass users and Control Women
MFI of DCF-DA (in arbitrary units)
LPG users
BMF users
Blood neutrophils, mean± SD 420.6 ±143.3 572.5± 136.5*
Blood monocytes, mean± SD 245.6±102.5 306.4± 124.8*
Blood lymphocytes, mean± SD 157.5± 72.7 179.4± 91.1**
Whole sputum, mean± SD 346.4± 188.1 708.8 ±274.9*
*p<0.0001 and **p< 0.05, compared with the LPG users in Students
Table 4. Spearmans Rank Correlation Analysis to Test an Association
Between Oxidative Stress and Markers and Mediators of Neutrophil
Neutrophil activation
With ROS With SOD
ρvalue pvalue ρvalue pvalue
CD16 0.516 0.048* 0.740 0.003*
CD35 0.601 0.021* 0.529 0.022*
CD11b 0.552 0.023* 0.378 0.041*
CD18 0.802 0.001* 0.508 0.021*
TNF-α0.543 0.037* 0.448 0.020*
IL-6 0.561 0.031* 0.396 0.038*
IL-12 0.112 0.124 0.291 0.102
IL-8 0.604 0.021* 0.683 0.008*
iNOS 0.681 0.012* 0.714 0.009*
NO 0.536 0.042* 0.385 0.040*
MPO 0.622 0.013* 0.462 0.016*
*p<0.05, considered as signicant
Table 5. Multivariate Logistic Regression Analysis of the Association
between Neutrophil Activation Markers and PM
and PM
Levels in
Indoor Air Controlling Potential Confounders
Neutrophil activation
With PM
With PM
OR 95% CI OR 95% CI
CD16 1.25 1.031.52 1.35 1.121.65
CD35 1.24 1.021.47 1.44 1.181.76
CD11b 1.33 1.121.56 1.68 1.351.96
CD18 1.37 1.151.63 1.64 1.252.13
TNF-α1.29 1.061.57 2.50 1.253.66
IL-6 1.24 1.041.64 1.89 1.662.23
IL-12 1.11 1.111.20 1.23 1.251.56
IL-8 1.37 1.151.63 1.64 1.252.13
iNOS 1.37 1.151.63 1.64 1.252.13
NO 1.33 1.121.56 1.68 1.351.96
MPO 1.37 1.151.63 1.64 1.252.13
All the associations were positive and statistically signicant
OR odds ratio, CI condence of intervals
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CD16 is widely expressed on the neutrophil surface,
and upregulation of this receptor contributes to disease
severity in sepsis [52]. On the other hand, CD35 is
responsible for controlling complement activation and the
binding of soluble immune complexes [33]. The engage-
ment of these receptors on the surface of neutrophils
stimulates neutrophil phagocytosis, degranulation, and
generation of ROS. Rise in blood leukocyte number,
increase in inammatory cells in spontaneously expecto-
rated sputa, overproduction of ROS, and depletion of SOD,
as observed in biomass-using women of this study, are
consistent with inammatory response [5355]. In addition,
increased expressions of neutrophil surface receptors CD16,
CD35, and CD11b/CD18 among biomass users may
suggest stimulation of the immune defense. CD11b on the
neutrophil surface plays an important role in the adhesion
and migration of neutrophils from circulation into the
airways and in the activation of neutrophils [17,56]. Apart
from its involvement in phagocytosis of opsonized bacteria,
CD11b/CD18 contributes to neutrophil aggregation and
adhesion to the endothelial surface [26]. Activated neutro-
phils express β
-integrin (CD11b/CD18) on their surface
because it is required for their transmigration into the area of
inammation. Neutrophil inux from blood to the airway
space is driven largely by IL-8, a potent neutrophil chemo-
attractant produced by airway epithelial cells. Biological
components of air pollution activate alveolar macrophages
to produce TNF-αwhich, in turn, stimulates airway
epithelial cells to produce IL-8. Therefore, upregulation of
CD11b/CD18 expression suggests accelerated migration of
neutrophils from the circulation to the tissues following
chronic exposures of the airways to biomass smoke. It is
important to mention in this context that smoking of tobacco
causes upregulation of CD11b/CD18 expression on neu-
trophil surface [57], increase in neutrophil retention in the
lung, and stimulation of granulocytemacrophage colony-
stimulating factor that can modulate neutrophil function
[58]. If the upregulation of CD11b/CD18 on neutrophil
surface is perpetuated, however, it can lead to adverse heath
outcome. For instance, augmented expression of neutrophil
CD11b is common among patients with COPD [59].
Neutrophils of biomass users also generated an excess of
MPO, suggesting neutrophil activation following cumula-
tive biomass smoke exposures. MPO, a heme protein, is a
critical mediator in coronary atherosclerosis [60]. The high
level of MPO in plasma due to the activation of neutrophils
is an early event in acute myocardial infarction and
potentially precedes myocardial injury [60].
Increase in the expression of iNOS in neutrophils
and remarkable elevation in serum NO are other
signicant observations among biomass users. NO is
an intracellular signal transmitter. Its excessive produc-
tion via iNOS and a subsequent oxidative stress reaction
are thought to be critically involved in the pathophysi-
ology of pulmonary sepsis [61]. Increase in iNOS
mRNA expression, iNOS activity, and NO secretion
have been recorded in alveolar macrophages in hyper-
oxia-induced lung injury in rat [62]. Conversely, sup-
pression of NO generation via downregulation of iNOS
by selective iNOS inhibitors protects from lung injury
The participants of this study were all never-
smokers and tobacco nonchewers, and exposure to
environmental tobacco smoke for the presence of
smoker in the family was similar among biomass and
LPG users. Therefore, it seems that neutrophilic inam-
matory changes among biomass-using women were not
due to tobacco smoking or chewing habit. The villages
where the participants resided were far from the high-
ways and busy road trafc. Bicycle and cycle rickshaw
were the principal mode of transport in these villages
and there were no air-polluting industries within 5 km
radius. Thus, ambient air pollution levels in the study
areas seemed negligible. Besides, biomass and LPG
users were neighbors, so the impact of outdoor air
pollution was similar in these two groups. Therefore, the
difference in indoor air quality between biomass- and
LPG-using households was perhaps responsible for the
inammatory changes among biomass users. But the
precise mechanism by which IAP elicits inammation is
currently unknown. Combustion of biomass generates
organic chemicals which become adsorbed on the outer
surface of the respirable PM and get entry inside the
respiratory tract through inhalation. Inorganic compo-
nent of the PM, especially the transitional metals, can
directly stimulate epithelial cells to produce IL-8 [65]
and consequent neutrophilic inammation in the lung
[66]. However, biomass smoke contains several other
pollutants such as volatile organic compounds toluene,
xylene, and benzene [67] which can elicit inammation
following inhalation [68].
Oxidative stress among biomass users, as evident
from the rise in ROS generation and depletion of SOD,
is another potential mechanism of inammation in these
women. Like biomass smoke, environmental tobacco
smoke and ambient (outdoor) air pollution also cause
oxidative stress and airway inammation [69,70].
Inhaled PM can induce pulmonary and systemic inam-
mation [71]resulting in ROS generation from inam-
matory cells. In essence, the present study shows that
680 Banerjee, Mondal, Das, and Ray
Author's personal copy
sustained exposures to biomass smoke during daily
household cooking cause the upregulation of circulating
neutrophil surface receptor expressions facilitating their
transmigration to the airways causing inammation in
women in their child-bearing age. Sustained inamma-
tion may cause tissue damage leading to various diseases
including COPD and cardiovascular illness. In view of
these possibilities of great public health concern, efforts
should be made to reduce IAP from biomass burning by
improving kitchen ventilation, by introducing improved
cook stoves, and nally by switching to cleaner fuel
The authors gratefully acknowledge the nancial
support received from Central Pollution Control Board,
Delhi in carrying out this study.
Conicts of interest statement. The authors declare
that there are no conicts of interest.
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683Neutrophilic Inammatory Response and Oxidative Stress
Author's personal copy
... They can decrease base-pairing error repair proteins in airway epithelial cells, which causes an increase in the incidence of cardiovascular-disease risk factors; endocrine diseases, such as diabetes and hypertension; female reproductive system diseases, such as infertility; and neuropsychiatric symptoms, such as anxiety [8,10,23]. Previous studies on Hb and the prevalence of anemia have shown an association between PM2.5, nitrogen dioxide (NO2), and access to blood (plasma and forming cell elements) [8,24]. The authors have argued that it makes them exceptionally vulnerable to the harmful effects of possible contaminants, such as lead and arsine, which damage the membranes of erythrocytes and cause anemia [7,25]. ...
... Furthermore, exposure to air pollution is significantly associated with increased anemia and decreased Hb levels in vulnerable groups [19]. This indicates that exposure to chronic air pollution is an important risk factor for anemia in vulnerable populations [19,24]. In addition, studies have proved the relationship between pollutants, increased oxidative stress, decreased blood folic acid, and anemia [9,22,31]. ...
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Exposure to indoor particulate matter (PM) is a potential risk factor that increases systemic inflammation and affects erythropoiesis. This study investigated the association between exposure to indoor PM and blood indicators related to anemia (BIRA) in housewives. Indoor PM and blood folate status are important factors in the risk of anemia. This was a housewife cohort study; we recruited 284 housewives in Seoul and Ulsan, Republic of Korea. Indoor exposure to PM2.5 and PM10 was measured by gravimetric analysis and sensors. We investigated the BIRA, such as hemoglobin (Hb), hematocrit, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), and mean corpuscular Hb concentration (MCHC). Statistical analysis was performed by multiple linear regression model and mediation analysis. The association between BIRA and PM was assessed by multiple linear regression models fitted by mediation analyses. The increase in the level of indoor PM2.5 was associated with a decrease in MCV (Beta coefficient (B): −0.069, Standard error (SE): 0.022) and MCH (B: −0.019, SE: 0.009) in gravimetric measurements. The increase in the level of indoor PM2.5 was associated with a decrease in Hb (B: −0.024, SE: 0.011), hematocrit (B: −0.059, SE: 0.033), and MCV (B: −0.081, SE: 0.037) and MCH (B: −0.037, SE: 0.012) in sensor measurements (PM2.5-Lag10). Further, we identified a serum folate-mediated PM effect. The indoor PM exposure was significantly associated with decreased Hb, MCV, and MCH in housewives. Taken together, our data show that exposure to indoor PM is a risk factor for anemia in housewives. Blood folate concentration can be a mediating factor in the effect of indoor PM on BIRA. Therefore, folate intake should be recommended to prevent anemia in housewives. Moreover, indoor PM exposure should be managed.
... 36,38,51 Few studies have investigated the relationship of HAP with markers of inflammation in the HAP literature. [52][53][54][55][56][57] These existing studies have evaluated the impact of different types of stoves on markers of inflammation or cardiovascular health, for example, comparing biomass stove use with clean fuel use [53][54][55][56][57] or comparing use of stoves with and without chimneys. 52 Furthermore, no HAP studies have explored the association of BC and inflammation markers in multi-pollutant models. ...
... 36,38,51 Few studies have investigated the relationship of HAP with markers of inflammation in the HAP literature. [52][53][54][55][56][57] These existing studies have evaluated the impact of different types of stoves on markers of inflammation or cardiovascular health, for example, comparing biomass stove use with clean fuel use [53][54][55][56][57] or comparing use of stoves with and without chimneys. 52 Furthermore, no HAP studies have explored the association of BC and inflammation markers in multi-pollutant models. ...
Household air pollution (HAP) from biomass stoves is a leading risk factor for cardiopulmonary outcomes; however, its toxicity pathways and relationship with inflammation markers are poorly understood. Among 180 adult women in rural Peru, we examined the cross‐sectional exposure‐response relationship between biomass HAP and markers of inflammation in blood using baseline measurements from a randomized trial. We measured markers of inflammation (CRP, IL‐6, IL‐10, IL‐1β, and TNF‐α) with dried blood spots, 48‐h kitchen area concentrations and personal exposures to fine particulate matter (PM2.5), black carbon (BC), and carbon monoxide (CO), and 48‐h kitchen concentrations of nitrogen dioxide (NO2) in a subset of 97 participants. We conducted an exposure‐response analysis between quintiles of HAP levels and markers of inflammation. Markers of inflammation were more strongly associated with kitchen area concentrations of BC than PM2.5. As expected, kitchen area BC concentrations were positively associated with TNF‐α (pro‐inflammatory) concentrations and negatively associated with IL‐10, an anti‐inflammatory marker, controlling for confounders in single‐ and multi‐pollutant models. However, contrary to expectations, kitchen area BC and NO2 concentrations were negatively associated with IL‐1β, a pro‐inflammatory marker. No associations were identified for IL‐6 or CRP, or for any marker in relation to personal exposures.
... Only a couple of recent nonrandomized pilot studies, with a similar LPG stove and behavioral interventions in India, have been able to achieve comparable reductions (Pillarisetti et al. 2019;Sambandam et al. 2020). Studies in Guatemala, Bangladesh, and India demonstrated PM indoor area reductions with LPG stove use in comparison with biomass use, which ranged from 45% to 75% (achieving concentrations between 50 to 800 lg=m 3 ) (Albalak et al. 2001;Banerjee et al. 2012;Begum et al. 2009;Dutta et al. 2012;Naeher et al. 2000). Most of these previous studies were crosssectional, some measured exposures on a small number of participants (Albalak et al. 2001;Begum et al. 2009;Naeher et al. 2000), and many participants continued to use both LPG and biomass stoves (Albalak et al. 2001;Gould et al. 2020;Naeher et al. 2000). ...
Background: Household air pollution (HAP) from biomass fuel combustion remains a leading environmental risk factor for morbidity worldwide. Objective: Measure the effect of liquefied petroleum gas (LPG) interventions on HAP exposures in Puno, Peru. Methods: We conducted a 1-y randomized controlled trial followed by a 1-y pragmatic crossover trial in 180 women age 25-64 y. During the first year, intervention participants received a free LPG stove, continuous fuel delivery, and regular behavioral messaging, whereas controls continued their biomass cooking practices. During the second year, control participants received a free LPG stove, regular behavioral messaging, and vouchers to obtain LPG tanks from a nearby distributor, whereas fuel distribution stopped for intervention participants. We collected 48-h kitchen area concentrations and personal exposures to fine particulate matter (PM) with aerodynamic diameter ≤2.5μm (PM2.5), black carbon (BC), and carbon monoxide (CO) at baseline and 3-, 6-, 12-, 18-, and 24-months post randomization. Results: Baseline mean [±standard deviation (SD)] PM2.5 (kitchen area concentrations 1,220±1,010 vs. 1,190±880 μg/m3; personal exposure 126±214 vs. 104±100 μg/m3), CO (kitchen 53±49 vs. 50±41 ppm; personal 7±8 vs. 7±8 ppm), and BC (kitchen 180±120 vs. 210±150 μg/m3; personal 19±16 vs. 21±22 μg/m3) were similar between control and intervention participants. Intervention participants had consistently lower mean (±SD) concentrations at the 12-month visit for kitchen (41±59 μg/m3, 3±6 μg/m3, and 8±13 ppm) and personal exposures (26±34 μg/m3, 2±3 μg/m3, and 3±4 ppm) to PM2.5, BC, and CO when compared to controls during the first year. In the second year, we observed comparable HAP reductions among controls after the voucher-based intervention for LPG fuel was implemented (24-month visit PM2.5, BC, and CO kitchen mean concentrations of 34±74 μg/m3, 3±5 μg/m3, and 6±6 ppm and personal exposures of 17±15 μg/m3, 2±2 μg/m3, and 3±4 ppm, respectively), and average reductions were present among intervention participants even after free fuel distribution stopped (24-month visit PM2.5, BC, and CO kitchen mean concentrations of 561±1,251 μg/m3, 82±124 μg/m3, and 23±28 ppm and personal exposures of 35±38 μg/m3, 6±6 μg/m3, and 4±5 ppm, respectively). Discussion: Both home delivery and voucher-based provision of free LPG over a 1-y period, in combination with provision of a free LPG stove and longitudinal behavioral messaging, reduced HAP to levels below 24-h World Health Organization air quality guidelines. Moreover, the effects of the intervention on HAP persisted for a year after fuel delivery stopped. Such strategies could be applied in LPG programs to reduce HAP and potentially improve health.
... An increase in NEUT and a decrease in LYM and MONO were independently associated with TAC levels. These parameters are predictors of inflammation, particularly in infection (Wu et al. 2021), and their association, particularly NEUT, with ROS generation has been documented (Banerjee et al. 2012). In a recent review, Goud et al. (2021) hypothesized that NEUT, eosinophils, MONO, macrophages, mitochondrial damage, and NADPH oxidase are the major sources of ROS generation at sites of inflammation. ...
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The role of micronutrient deficiency in the pathogenesis of COVID-19 has been reviewed in the literature; however, the data are limited and conflicting. This study investigated the association between the status of essential metals, vitamins, and antioxidant enzyme activities in COVID-19 patients and disease severity. We recruited 155 patients, who were grouped into four classes based on the Adults guideline for the Management of Coronavirus Disease 2019 at King Faisal Specialist & Research Centre (KFSH&RC): asymptomatic (N = 16), mild (N = 49), moderate (N = 68), and severe (N = 22). We measured serum levels of copper (Cu), zinc (Zn), selenium (Se), vitamin D3, vitamin A, vitamin E, total antioxidant capacity, and superoxide dismutase (SOD). Among the patients, 30%, 25%, 37%, and 68% were deficient in Se (< 70.08 µg/L), Zn (< 0.693 µg/mL), vitamin A (< 0.343 µg/mL), and vitamin D3 (< 20.05 µg/L), respectively, and SOD activity was low. Among the patients, 28% had elevated Cu levels (> 1.401 µg/mL, KFSH&RC upper reference limit). Multiple regression analysis revealed an 18% decrease in Se levels in patients with severe symptoms, which increased to 30% after adjusting the model for inflammatory markers. Regardless of inflammation, Se was independently associated with COVID-19 severity. In contrast, a 50% increase in Cu levels was associated with disease severity only after adjusting for C-reactive protein, reflecting its possible inflammatory and pro-oxidant role in COVID-19 pathogenesis. We noted an imbalance in the ratio between Cu and Zn, with ~ 83% of patients having a Cu/Zn ratio > 1, which is an indicator of inflammation. Cu-to-Zn ratio increased to 45% in patients with mild symptoms and 34%–36% in patients with moderate symptoms compared to asymptomatic patients. These relationships were only obtained when one of the laboratory parameters (lymphocyte or monocyte) or inflammatory markers (neutrophil-to-lymphocyte ratio) was included in the regression model. These findings suggest that Cu/Zn might further exacerbate inflammation in COVID-19 patients and might be synergistically associated with disease severity. A 23% decrease in vitamin A was seen in patients with severe symptoms, which disappeared after adjusting for inflammatory markers. This finding may highlight the potential role of inflammation in mediating the relationship between COVID-19 severity and vitamin A levels. Despite our patients’ low status of Zn, vitamin D3, and antioxidant enzyme (SOD), there is no evidence of their role in COVID-19 progression. Our findings reinforce that deficiency or excess of certain micronutrients plays a role in the pathogenesis of COVID-19. More studies are required to support our results.
... Subsequently, pathological changes in COPD include airflow limitation, abnormal gas exchange, and decreased lung function. [5][6][7][8] Therefore, an in-depth study of the specific mechanisms underlying airway inflammation in COPD is of great significance for the diagnosis and treatment of COPD. ...
Full-text available
Purpose: Chronic obstructive pulmonary disease (COPD) is associated with a complex inflammatory regulatory network. Resistin-like molecule β (RELMβ) is highly expressed in the lungs of COPD patients. We aimed to investigate the proinflammatory effect of RELMβ on airway epithelial cells in COPD. Methods: First, a GEO dataset was used to analyze the expression of the RELMβ gene in the COPD and control groups as well as the protein levels of RELMβ in the sera of outpatients with COPD and normal control subjects in our hospital. We also stimulated 16HBE bronchial epithelial cells with recombinant RELMβ protein and analyzed the expression of IL-8 and IL-1β. We upregulated and downregulated the gene expression of RELMβ in 16HBE cells and analyzed the expression of the inflammatory cytokines IL-8 and IL-1β. In addition, we also examined the mechanism by which the p38 MAPK signaling pathway contributed to the regulation of IL-8 and IL-1β expression by RELMβ. Results: RELMβ expression was increased in COPD tissues in different data sets and in the serum of COPD patients in our hospital. IL-8 and IL-1β expression was also increased in COPD tissues with high RELMβ gene expression in different data sets. The RELMβ gene was mainly related to inflammatory factors and inflammatory signaling pathways in the PPI regulatory network. Experiments at the cellular level showed that RELMβ promoted the expression of the inflammatory cytokines IL-8 and IL-1β, and this regulation was mediated by the p38 MAPK signaling pathway. Conclusion: RELMβ can promote the expression of the inflammatory cytokines IL-8 and IL-1β in bronchial epithelial cells of patients with COPD and exert inflammatory effects. RELMβ may be a potential target for the treatment of COPD.
... These findings suggest persistent inflammation in response to air pollution-induced oxidative stress. Cooking with biomass has also been reported to alter sputum cytology (increasing counts of neutrophils, lymphocytes, eosinophils and alveolar macrophages (AM)) increase airway inflammation (higher sputum levels of IL6, 8 and TNFα) and oxidative stress (enhanced ROS generation and depletion of SOD activity) that might result in further amplification of the tissue damaging cascade in women chronically exposed to biomass smoke (Banerjee et al. 2012;Dutta et al. 2013). Prevalence of mucus plugs, goblet cell hyperplasia, and nuclear anomaly of columnar epithelial cells was found to be higher in urban subjects exposed to high levels of urban air pollution in Kolkata as compared to controls drawn from relatively cleaner peri-urban zones (Ray and Lahiri 2010). ...
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Air pollution ranks among the leading risk factors contributing to the burden of disease in South Asia with household and ambient air pollution accounting for 6 percent and 3 percent respectively of the total national burden of disease in India. Both urban and rural communities bear this burden in terms of premature mortality and disability-adjusted life years, resulting from excess risks of communicable and non-communicable diseases. We review the information pertaining to exposures to fine particulate matter and air toxics together with the attributable disease burden estimates. We also provide a summary of the results from recent assessments on carcinogenicity of ambient and household air pollution conducted by The International Agency for Research on Cancer. We conclude with a list of specific priorities for action related to air toxics and cancer in India.
... Human Body over South Asia A few studies from India investigated the role of PM exposure on the health of biological tissues using a variety of markers listed in Table 1 (Das et al., 2021;Jan et al., 2020;Roy et al., 2015;Sambandam et al., 2015;Dutta et al., , 2012Banerjee et al., 2012;Mondal et al., 2011; Rahman et al., 1997;Arif et al., 1992). Here, Arif et al. (1992) and Rahman et al. (1997) investigated the toxic potential of various particles and fibers to the human and rat alveolar macrophages. ...
Full-text available
South Asia occupies only about 3.5% of the world’s area but, about 25% of the average world’s population lives here and is continuously exposed to severe air pollution. Unprecedented development activities in most of the South Asian cities emit primary and secondary pollutants into the atmosphere. Particulate matter (PM), a principal air pollutant, are tiny enough to remain suspended in the atmosphere for a long time (about a week). They can penetrate the human nasal airway and damage the lungs. PM effects on human health are assessed based on their mass concentration, size distribution, and chemical composition. Despite being critically important, studies related to PM effects on human health are limited over South Asia. In recent years, only a few South Asian research groups started studying the ability of atmospheric PM to cause human health hazards by generating in situ reactive oxygen species (ROS). The capability of atmospheric PM to produce ROS and/or deplete antioxidants is termed as their oxidative potential (OP). Though limited, efforts are made to identify particular species with the higher OP. Atmospheric aging of PM can also alter their OP. No studies from South Asia, except a few from India, investigated how the atmospheric aging changes the chemical and physical properties of PM and affect their OP over South Asia. These studies also showed that OP depends more on PM composition rather than its concentrations. Therefore, mitigation strategies for reducing PM mass concentrations alone may not be sufficient, and linking PM OP with significant health effects may be a better way to regulate specific sources of PM rather than overall PM mass. This review reports the necessities and limitations for PM OP studies in South Asia and future directions.
... IL-1β is shown to be associated with neutrophilic inflammation of the airways. Sputum examination of biomass smoke exposed women show greater neutrophilic inflammation as compared to clean fuel users with evidence of neutrophilic activation [44]; circulating neutrophils show an increased surface expression of CD16 (F C γ receptor III), β 2 Mac-1 integrin (CD11b/CD18) and CD35 (complement receptor-1), double the levels of neutrophil chemoattractant cytokine IL-8, higher myeloperoxidase activity from circulating neutrophils, and that inducible nitric oxide synthase (iNOS) was expressed more in neutrophils from airways of biomass exposed women, who also had double the concentration of serum nitric oxide. ...
Full-text available
Chronic obstructive pulmonary disease (COPD), the leading cause of mortality and morbidity worldwide, is characterized by abnormal activation of inflammatory cells. The increased pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β), further amplify the inflammation. We evaluated the dose response relationship of IL-1β and TNF-α levels and severity of airflow limitation, and differential responses in IL-1β and TNF-α between biomass COPD (BMS-COPD) and tobacco smoke COPD (TS-COPD) using a case control design in 160 subjects. Patients with COPD had higher serum levels of both IL-1β and TNF-α compared to healthy controls. A large difference in TNF-α was observed between TS-COPD and BMS-COPD, where TS-COPD patients had much higher levels. Serum IL-1β levels were higher in BMS-COPD. Levels of IL-1β correlated better with severity of airflow limitation than TNF-α levels. Both TNF-α and IL-1β levels had a negative linear relationship with Forced Expiratory Volume in 1st second (FEV1) and six-minute walk distance. The correlations were stronger with FEV1 than six-minute walk distance. The correlations of TNF-α and IL-1β with St George Respiratory Questionnaire (SGRQ) scores and body mass index (BMI) were not significant. In conclusion, the levels of pro-inflammatory cytokines TNF-α and IL-1β are differently elevated in TS-COPD and BMS-COPD, respectively.
... lung cancer, chronic obstructive pulmonary disease) and cardiovascular diseases (e.g. stroke, heart disease) [10][11][12][13][14][15][16]. The Global Burden of Disease (GBD) study attributed a total of 3.6 million deaths annually from exposure to household air pollution (HAP) in 2019, making it the 2nd highest environmental risk factor [17]. ...
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This longitudinal study presents the joint effects of a COVID-19 community lockdown on household energy and food security in an informal settlement in Nairobi, Kenya. Randomly administered surveys were completed from December 2019-March 2020 before community lockdown (n = 474) and repeated in April 2020 during lock-down (n = 194). Nearly universal (95%) income decline occurred during the lockdown and led to 88% of households reporting food insecurity. During lockdown, a quarter of households (n = 17) using liquefied petroleum gas (LPG), a cleaner cooking fuel typically available in pre-set quantities (e.g. 6 kg cylinders), switched to polluting cooking fuels (kerosene, wood), which could be purchased in smaller amounts or gathered for free. Household size increases during lockdown also led to participants' altering their cooking fuel, and changing their cooking behaviors and foods consumed. Further, households more likely to switch away from LPG had lower consumption prior to lockdown and had suffered greater income loss, compared with households that continued to use LPG. Thus, inequities in clean cooking fuel access may have been exacerbated by COVID-19 lockdown. These findings demonstrate the complex relationship between household demographics, financial strain, diet and cooking patterns, and present the opportunity for a food-energy nexus approach to address multiple Sustainable Development Goals (SDGs): achieving zero hunger (SDG 2) and universal affordable, modern and clean energy access (SDG 7) by 2030. Ensuring that LPG is affordable, accessible and meets the dietary and cooking needs of families should be a policy priority for helping improve food and energy security among the urban poor.
Household air pollution (HAP) arising from combustion of biomass fuel (BMF) is a leading cause of morbidity and mortality in low-income countries. Air pollution may stimulate pro-inflammatory responses by activating diverse immune cells and cyto/chemokine expression, thereby contributing to diseases. We aimed to study cellular immune responses among women chronically exposed to HAP through use of BMF for domestic cooking. Among 200 healthy, non-smoking women in rural Bangladesh, we assessed exposure to HAP by measuring particulate matter 2.5 (PM2.5), black carbon (BC) and carbon monoxide (CO), through use of personal monitors RTI MicroPEM™ and Lascar CO logger respectively, for 48 h. Blood samples were collected following HAP exposure assessment and were analyzed for immunoprofiling by flow cytometry, plasma IgE by immunoassay analyzer and cyto/chemokine response from monocyte-derived-macrophages (MDM) and -dendritic cells (MDDC) by multiplex immunoassay. In multivariate linear regression model, a doubling of PM2.5 was associated with small increments in immature/early B cells (CD19+CD38+) and plasmablasts (CD19+CD38+CD27+). In contrast, a doubling of CO was associated with 1.20% reduction in CD19+ B lymphocytes (95% confidence interval (CI) = -2.36, -0.01). A doubling of PM2.5 and BC each was associated with 3.12% (95%CI = -5.85, -0.38) and 4.07% (95%CI = -7.96, -0.17) decrements in memory B cells (CD19+CD27+), respectively. Exposure to CO was associated with increased plasma IgE levels (beta(β) = 240.4, 95%CI = 3.06, 477.8). PM2.5 and CO exposure was associated with increased MDM production of CXCL10 (β = 12287, 95%CI = 1038, 23536) and CCL5 (β = 835.7, 95%CI = 95.5, 1576), respectively. Conversely, BC exposure was associated with reduction in MDDC-produced CCL5 (β = -3583, 95%CI = -6358, -807.8) and TNF-α (β = -15521, 95%CI = -28968, -2074). Our findings suggest that chronic HAP exposure through BMF use adversely affects proportions of B lymphocytes, particularly memory B cells, plasma IgE levels and functions of antigen presenting cells in rural women.
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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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Objective: To study the status of oxidative stress and DNA damage repair in circulating blood leukocytes of heart failure patients supported by continuous flow left ventricular assist devices (LVADs). Materials and methods: Ten HF patients implanted with LVAD as bridge to transplant or destination therapy were enrolled in the study and 10 age and sex matched volunteers were recruited as the study control. Reactive oxygen species (ROS) in blood leukocytes and superoxide dismutase (SOD) in erythrocytes were measured by flow cytometry/immunofluorescence microscopy and spectrophotometry, respectively. ELISA was used to measure oxidized low density lipoproteins (oxLDL) in plasma. Markers of DNA damage (γ-H2AX) and repairs (Mre11, DNA ligase IV, Ku70, and Ku80) were quantified in blood lymphocytes by immunocytochemistry. Results: Levels of ROS and oxLDL were significantly higher in HF patients with LVAD than baseline as well as the control group; moreover, SOD levels were decreased with increasing post-operative periods. All the changes indicated enhanced oxidative stress among LVAD recipients. Significantly higher γ-H2AX foci in lymphocytes confirmed DNA double strand breaks in LVAD recipients. γ-H2AX foci numbers in lymphocytes were positively correlated with the ROS and oxLDL and negatively with SOD levels (p<0.0001). Expressions of DNA ligase IV, Ku70 and Ku80 proteins were highest after one week and Mre11 protein after 3 months of LVAD transplantation; indicated abnormal DNA repair. Conclusions: The study, for the first time shows that, continuous flow LVAD implanted HF patients not only exhibit elevated oxidative stress and DNA damage in blood leukocytes but also have abnormalities in DNA repair pathways.
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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.
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Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood. We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis. The in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke. Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.
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Superoxide dismutase (EC has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.
Study objectives: To identify the characteristics of airway inflammation in persistent asthma and to examine the role of neutrophilic inflammation in noneosinophilic persistent asthma. Methods: Nonsmoking adults (n = 56) with persistent asthma and healthy control subjects (n = 8) underwent hypertonic saline solution challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for total cell count, cellular differential, supernatant eosinophil cationic protein (ECP), myeloperoxidase, and interleukin (IL)-8. Results: We identified two distinct inflammatory patterns. Typical eosinophilic inflammation occurred in 41% of subjects, whereas the remainder exhibited noneosinophilic asthma (59%). Both neutrophil percentage and absolute neutrophil counts were increased in subjects with noneosinophilic asthma (64%, 283 × 106/mL) compared to eosinophilic asthma (14%, 41 × 106/mL) and control subjects (34%, 49 × 106/mL; p = 0.0001). Myeloperoxidase was elevated in both noneosinophilic (280 ng/mL) and eosinophilic groups (254 ng/mL) compared with control subjects (82 ng/mL; p = 0.002). Sputum IL-8 levels were highest in subjects with noneosinophilic asthma (45 ng/mL) compared to eosinophilic asthma (9.6 ng/mL) and control subjects (3.5 ng/mL; p = 0.0001). Neutrophils correlated with IL-8 levels (r = 0.72). ECP was highest in subjects with eosinophilic asthma (2,685 ng/mL) compared with noneosinophilic asthma (1,081 ng/mL) and control subjects (110 ng/mL; p = 0.0001). Conclusion: Induced-sputum analysis in persistent asthma identifies two different inflammatory patterns. The most common pattern is noneosinophilic, associated with a neutrophil influx and activation, which may be mediated by IL-8 secretion. There is heterogeneity of airway inflammation in persistent asthma, which indicates differing mechanisms and may impact on treatment responses.
Background: Epidemiological research and meta-analyses of published data have shown that biomass smoke (BS) is a risk factor for chronic obstructive pulmonary disease (COPD). However, the link between BS and COPD lacks experimental confirmation. Objectives: To verify whether BS can induce pathologic changes and systemic oxidative stress, which may be relevant to the development of emphysema and chronic bronchitis in rats. Methods: Rats were exposed to BS, cigarette smoke (CS), or clean air (sham) for 14 weeks. During the exposure, the O2, SO2, and CO levels were monitored. Pathological changes in the lungs, systemic oxidative stress, and inflammation biomarkers, together with GSTM1 and GSTP1 mRNA expression in the lung were measured. The glutamate-cysteine ligase catalytic subunit (GCLC) protein expression in the lung was measured using immunohistochemistry and western blotting. Results: The O2, CO, and SO2 levels were 20.31 ± 0.03%, 981.72 ± 64.76, and 2.59 ± 0.26 mg/m(3) for the BS group, respectively, while their levels in the CS group were 20.28 ± 0.15%, 745.56 ± 30.83, and 12.64 ± 0.591 mg/m(3) respectively. As with the rats exposed to CS, the BS rats showed an increased number of inflammatory cells in the bronchoalveolar lavage fluid, an increased pulmonary mean linear intercept and a decreased pulmonary mean alveolar number. Characteristics of chronic bronchitis and peribronchial fibrosis were also found in the BS-exposed rat lungs. Reduced body weight, systemic oxidative stress, and increased GCLC protein expression in the lungs were observed in the rats exposed to BS and CS. Conclusions: BS can cause emphysema and chronic bronchitis similar to that caused by CS, which is accompanied by systemic oxidative stress and inflammation.