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Upregulation of AgNOR Expression in Epithelial Cells and Neutrophils in the Airways and Leukocytes in Peripheral Blood of Women Chronically Exposed to Biomass Smoke

Authors:

Abstract

To evaluate the impact of indoor air pollution from biomass fuel use on ribosome biogenesis in airway cells and peripheral blood leukocytes using the argyrophilic nucleolar organizer region (AgNOR) staining technique. Biomass users were represented by 78 never-smoking, premenopausal women from rural India and a control group of 73 age-matched women who cooked with liquefied petroleum gas (LPG). For silver staining, exfoliated airway cells and circulating lymphocytes and neutrophils were obtained from expectorated sputum and venous blood smears, respectively. Particulate pollution in indoor air was measured by real-time aerosol monitor. Compared with the controls, a statistically significant increase was observed in mean number of AgNOR dots per nucleus, their size, and the percentage of NOR-occupied nuclear area in exfoliated airway epithelial cells, airway neutrophils, and circulating lymphocytes and neutrophils of biomass users. Biomass-using households had 2 to 4 times more particulate pollutants than that of LPG-using households; the changes in AgNOR expression, especially in proliferating basal cells, were positively associated with PM10 and PM2.5 levels in indoor air after controlling potential confounders such as age, kitchen location, and family income. Indoor air pollution from biomass fuel use upregulates ribosome biogenesis in both the airways and peripheral blood.
An official Periodical of
The International Academy of Cytology
and the
Italian Society of Urologic Pathology
Upregulation of AgNOR Expression in Epithelial
Cells and Neutrophils in the Airways and
Leukocytes in Peripheral Blood of Women
Chronically Exposed to Biomass Smoke
Nandan Kumar Mondal, M.Sc., Debangshu Das, M.Sc., Bidisha Mukherjee,
M.Sc., and Manas Ranjan Ray, Ph.D.
From the Department of Experimental Hematology, Chittaranjan National Cancer Institute, Kolkata, India.
Messrs. Mondal, Das, and Mukherjee are Research Fellows.
Dr. Ray is Senior Scientific Officer and Head.
Supported by the Central Pollution Control Board, Delhi, India.
Address correspondence to: Manas Ranjan Ray, Ph.D., Department of Experimental Hematology, Chittaranjan National Cancer Institute,
37, S. P. Mukherjee Road, Kolkata 700 026, India (manasrray@rediffmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.
0884-6812/11/3301-0050/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytology and Histology® 50
ANALYTICAL
AND
QUANTITATIVE
CYTOLOGY AND
HISTOLOGY
®
OBJECTIVE: To evaluate the impact of indoor air
pollution from biomass fuel use on ribosome
biogenesis in airway cells and peripheral blood
leukocytes using the argyrophilic nucleolar organizer
region (AgNOR) staining technique.
STUDY DESIGN: Biomass users were represented
by 78 never-smoking, premenopausal women from
rural India and a control group of 73 age-matched
women who cooked with liquefied petroleum gas
(LPG). For silver
staining, exfoliated airway ce
lls
and circulating lymphocytes and neutrophils were
obtained from expectorated sputum and venous blood
smears, respectively. Particulate pollution in indoor
air was measured by real-time aerosol monitor.
RESULTS: Compared with the controls, a
statistically significant increase was observed in
mean number of AgNOR dots per nucleus, their size,
and the percentage Biomass-using households had 2
Mondal et al
Analytical and Quantitative Cytology and Histology®
to 4
-
times more particulate pollutants th
an that of
LPG-using households and the changes in AgNOR
expression especially in proliferating basal cells were
positively associated with PM
10
and PM
2.5
levels in
indoor air after controlling potential confounders like
age, location of the kitchen and family income.
CONCLUSION: Indoor air pollution from biomass
fuel use upregulates ribosome biogenesis both in the
airways and in peripheral blood. (Anal Quant Cytol
Histol 2011; 33:50–59)
Keywords: AgNOR, airway cells, blood
leukocytes, biomass fuel, women.
Indoor air pollution (IAP) from smoke emitted
during cooking with unprocessed, solid biomass
is a major health concern in the developing
world
1
. Approximately half of the world’s
population and up to 90% of households in rural
areas of Asia, sub-Saharan Africa and Latin
America still depend on biomass such as wood,
dung and crop residues for domestic cooking
and room heating
2
. Biomass users are mostly
poor people who cannot afford cleaner fuels and
they usually burn these fuels in traditional
stoves that are not vented outside. Incomplete
combustion of biomass coupled with lack of
smoke outlet often results in very high level of
IAP. In a typical Indian household, for example,
the concentration of PM
10
(particulate matter
having aerodynamic diameter of less than 10
µm) during cooking with biomass varies
between 500 and 2000 µg/m
3 3
, which is several
times higher than the air quality standard
recommended by the US Environmental
Protection Agency
4
.
Biomass smoke is essentially not different
from cigarette smoke. It contains a large number
of health-damaging chemicals including high
level of particulate matter of different sizes,
carbon monoxide, oxides of nitrogen,
formaldehyde, acrolein, benzene, toluene,
styrene, 1,3-butadiene, and polycyclic organic
hydrocarbons including benzo (a) pyrene
5
and
transitional metals like Cu, Fe, Ni, Al and Zn
6,7
.
Some of the biomass smoke constituents like the
volatile organic compound benzene and
polycyclic aromatic hydrocarbon benzo (a)
pyrene are mutagenic and carcinogenic, and the
International Agency for Research on Cancer
(IARC) has categorized biomass smoke as a
‘probable carcinogen to humans’ (Group 2a)
8
.
Studies conducted in India and Mexico has
revealed a link between biomass smoke
exposure and the development of
adenocarcinomas of the lung in nonsmoking
women who cook with these fuels
9,10
. It is
reasonable to assume therefore that chronic
exposures to biomass smoke may cause
cytopathological changes particularly in those
cells that are at the direct line of exposure, such
as cells of the nasopharynx, oral cavity, airways
and the lung. However, little information is
available regarding cellular response of the
airways and peripheral organs to chronic
biomass smoke exposures. In view of this, we
have investigated in this study the impact of
chronic inhalation of biomass smoke on
ribosome biogenesis in epithelial and
inflammatory cells of the airways and
leukocytes in peripheral blood. For this, we
undertook the argyrophilic nucleolar organizer
regions (AgNOR) cytochemical assay in
exfoliated airway epithelial cells and neutrophils
in expectorated sputum and neutrophils and
lymphocytes in peripheral blood of rural
women who used to cook exclusively with
biomass fuel.
AgNORs are structural-functional units of the
nucleolus in which all the components necessary
for the biogenesis of ribosomal RNA (rRNA) are
located. They represent loops of DNA on the
short arms of acrocentric chromosomes that are
actively transcribing to rRNA and then to
ribosomes and finally to protein
11-13
. The
nucleolar organizer regions (NORs) are
associated with several non-histone proteins
that are visualized as black dots when stained
with silver (Ag). Accordingly the cytochemical
technique is known as AgNOR
14,15
. During
interphase, the main AgNOR proteins are
nucleolin (C23) and nucleophosmin (B23) which
Volume 33, Number 1/February 2011
AgNOR Expression in Biomass Smoke Exposure
52
are involved in ribosome biogenesis
16
, whereas
NORs contain subunits of RNA polymerase-I
and transcription factor UBF during mitosis
17
.
Intensity of AgNOR staining correlates
positively with the amount of these proteins
16
,
and the number of interphase AgNORs
correlates well with rRNA transcriptional
activity and, in continuously proliferating cells,
with the rapidity of cell proliferation
18
. AgNOR
staining is particularly important for
investigating the process of carcinogenesis in the
airways, as increasing numbers and areas of
AgNORs have been found in atypical squamous
epithelial cell, early squamous cell carcinoma
and invasive squamous cell carcinoma
19
.
In view of these reports, we employed
AgNOR method to assess the impact of
cumulative biomass smoke exposure on
ribosome biogenesis and the consequent risk of
carcinogenesis in the airways of never-smoking
premenopausal women from rural India who
used to cook with biomass for the past five years
or more and have compared the findings with
that of age-matched control women from same
locality who cooked with cleaner fuel liquefied
petroleum gas (LPG).
Methods
Participants
A total number of 151 pre-menopausal women
aged between 23 and 43 years from some
villages of Burdwan, Birbhum and Hooghly
districts of West Bengal, a state in eastern India,
volunteered to participate in this study. They
attended medical camps organized in their
villages. Among the participants, 78 women
(age 23 – 43 yr, median 34 yr) used to cook daily
for 2 – 6 hours exclusively with wood, cow dung
and agricultural wastes, such as bamboo, jute
stick, paddy husk, hay, dried leaves, etc.
Accordingly they were grouped as biomass
users. The remaining 73 women aged between
24 and 43 yr, median age 33 yr from same
villages used to cook with cleaner fuel LPG, and
they were considered as control.
Inclusion
and
E
xclusion
C
riteria
The inclusion criteria were apparently healthy,
tobacco non-smoking and non-chewing pre-
menopausal married women actively engaged
in daily household cooking for the past 5 years
or more and having a body mass index > 15 and
< 30 kg/m
2
. Pregnant women, those using oral
contraceptives, had history of malignancy or
were currently under medication were
excluded.
Questionnaire Survey for Collection of Background
Data
During personal interview with female
members of the research team, each participant
was requested to provide information about age,
education, family size and income, habit,
cooking time per day, years of cooking, fuel and
oven type, location of kitchen, health problems
in past 3 moths and last one 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, Kolkata approved the study
protocol.
Collection of Expectorated Sputum and Blood
Samples
Each participant was given a sterile plastic cups
to collect the early morning sputum after rinsing
her mouth with sterile normal saline to remove
extraneous material. Samples were collected for
three consecutive days to get representative
sample from the lower airways
20
. Two smears
were prepared on clean glass slides from the
non-transparent highly viscous parts of each
sputum sample. The slides were air dried and
fixed immediately with Carnoy’s solution
(ethanol: glacial acetic acid, 3 : 1, v/v) for 20 min
at the site of sample collection.
Blood (0.5 ml) was collected from the
antecubital venous plexus after informed
consent
Mondal et al
Analytical and Quantitative Cytology and Histology®
consent on the third day of sputum collection at
a fixed time (9:30-10:30 AM). For each
participant, two thin blood smears were
prepared on clean glass slides from venous
blood that was not mixed with anticoagulant.
AgNOR Staining of Sputum Cells
AgNOR staining was performed following the
procedure of Ploton et al (1986)
14
. In brief, slides
were incubated in darkness for 30 min at 37
0
C in
a colloidal silver solution containing 1 volume of
2% gelatin in 1% formic acid and 2 volumes of
50% aqueous silver nitrate solution. Thereafter
the slides were washed with distilled water,
fixed in 5% sodium thiosulfate for 5 min,
dehydrated in acetone with three changes 15
min each, cleared in xylene and mounted with
distrene plasticizer xylene (DPX).
AgNOR Staining in Blood Cells
AgNOR staining of blood film was done
following the procedure of Lindner (1993)
21
with
some modifications. In brief, the air-dried slides
were fixed in absolute methanol for 7 min at
room temperature. Then 250 μl of colloidal
silver solution containing 1 volume of 2%
gelatin in 1% formic acid and 2 volumes of 50%
aqueous silver nitrate solution was poured over
the slides and incubated at 37
0
C for 20 min.
Thereafter the slides were rinsed vigorously in
distilled water, air- dried and mounted with
glycerin.
Scoring and Image Analysis of AgNORs Stained
Slides
The slides were coded and examined under light
microscope (Leica DM1000 equipped with Leica
EC3 camera, Germany) using 100x oil
immersion lens. AgNOR scoring was restricted
to airway epithelial cells and neutrophils in
sputa and neutrophils and lymphocytes in blood
film for their relative abundance. For each cell
type, an average of 300 non-overlapping cells
was screened. The mean number of AgNORs
per nucleus, area of individual dot (in μm
2
) and
percentage of nuclear area occupied by AgNOR
dots were calculated by means of the computer-
assisted image analysis system (Lasez, version
1.5.0; Leica Microsystem limited, Switzerland).
Measurements of PM
10
and PM
2.5
in Indoor Air
Particulate matter with aerodynamic diameter
less than 10µm (PM
10
) and 2.5µm (PM
2.5
) were
measured in the cooking areas with real-time
laser photometer (DustTrakTM Aerosol
Monitor, model 8520, TSI Inc., MN, USA) that
contained 10-mm nylon Dor-Oliver cyclone and
operated at a flow rate of 1.7 lit/min, measuring
particle load in the concentration range of 1µg to
100mg/m
3
. The monitor was calibrated to the
standard ISO 12103-1 A1 test dust. Since
biomass-using women cook in a sitting position
2 - 3 ft away from the open chullah (oven), the
monitor was placed in the breathing zone of the
cook, 2.5 ft above 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 placed accordingly at a height of
4.5 ft.
Statistical Analysis
Statistical significance of the differences in
different parameters between biomass users and
control group was determined by Chi-square
test, Mann Whitney U test and Student’s ‘t’ test,
as applicable. Statistical analyses of collected
data were done using EPI info 6 and SPSS
statistical software (Statistical Package for Social
Sciences for windows, release 10.0, SPSS Inc.,
Chicago, USA) and p < 0.05 was considered
significant.
Results
Demographic and Socioeconomic Characteristics
Demographic and socio-economic characteristics
of study population are summarized in Table I.
Table I Demographic and Socioeconomic Characteristics of the Participants
Variable LPG-users (n = 73) Biomass user (n = 78) p value*
Age in yr, median (range) 33 (24 - 43) 34 (23 - 43) NS
Me
dian body mass index in kg/m
2
(range)
24.4 (
23.7
-
25.2
)
24.1 (23.3
-
25.4)
Cooking years, median (range)
14 (5
-
21)
15 (5
-
24
)
Cooking hours per day, median (range)
3.0 (2.0
5.0)
3.0 (2.5
5.5)
Years of schooling, median (range)
8 (0
-
14)
3
(0
-
9)
<0.05
Homes with separate kitchen (%)
90.4
59.0
< 0.05
Smoker in family (%)
43.8
44.9
Use of mosquito repellant at home (%)
74.5
72.5
Food habit, mixed (%)
95.9
96.1
Members in family, median (range)
4 (2
-
6)
4 (2
-
7)
Average fa
mily income per month
(in US $)
78
42
< 0.05
*Analyzed by χ2 test.
NS, statistically not significant.
Volume 33, Number 1/February 2011
AgNOR Expression in Biomass Smoke Exposure
54
LPG-using control group and biomass using
women were comparable with respect to age,
body mass index, cooking time, smoking and
chewing habit. But they differed significantly
with respect to education, presence of separate
kitchen and family income (p < 0.05 in Chi-
square test).
Particulate Pollutants in Indoor Air
The 8-hr mean concentration of PM
10
in indoor
air of biomass-using kitchen was 249 ± 143 (SD)
μg/m
3
in contrast to 73 ± 42 μg/m
3
in LPG-
using households ( p<0.001). The corresponding
PM
2.5
levels were 132 ± 75 μg/m
3
and 40 ± 29
μg/m
3
in biomass and LPG-using households,
respectively (p<0.001).
AgNOR Expression in Cells Present in Expectorated
Sputum
AgNORs are seen as blackish brown dots within
the nuclei. The number and size of AgNORs
differed with the cell type (Fig. 1, 2). Compared
with LPG-using control women, biomass users
had significantly increased number of AgNOR
dots per nucleus of basal, parabasal and
intermediate epithelial cells present in
expectorated sputum (p<0.0001; Table II).
Besides, individual AgNOR dots were larger in
size in biomass users (Fig. 1).
As a consequence, the percentage of nuclear
area occupied by AgNOR was significantly
higher in biomass-using women when
compared with that of controls (p<0.0001, Table
II). Like the epithelial cells, neutrophils present
in sputum samples of biomass-using women
had more AgNOR dots per nucleus and the dots
were larger in size and occupied greater nuclear
area than airway neutrophils of control women
(Fig.1, Table II).
AgNOR Expression in Peripheral Blood
Lymphocytes and Neutrophils
The number of AgNOR dots in peripheral blood
lymphocytes and neutrophils was more in
biomass using women, but the change was not
Figure 1 Photomicrographs of sputum cells showing nuclear localization of AgNOR dots in (a and b) basal
epithelial cells and (c and d) airway neutrophils. Among biomass users, the number and size of AgNOR dots in (b)
epithelial cells and (d) neutrophils were significantly elevated compared to that of LPG-using controls (a and c)
(AgNOR staining, × 1,000; inset × 1,500 (a,b), x3,000 (c,d).
Mondal et al
Analytical and Quantitative Cytology and Histology®
statistically significant (p = 0.0950 and p =
0.0575, respectively). The size of the AgNOR
dots and nuclear area occupied by these dots,
however, were significantly higher (p<0.0001) in
peripheral blood lymphocytes and neutrophils
of biomass users than their LPG-using neighbors
(Fig. 2, Table II).
Association between AgNOR Expression and
Cooking Years
A strong positive correlation was found in
Spearman’s rank correlation test between the
number of AgNOR dots per nucleus of airway
epithelial cells (r = 0.9965, p<0.0001) and airway
neutrophils (r = 0.9865, p<0.0001) and years of
cooking with biomass (Fig. 3a). Moreover,
cooking years with biomass was positively
associated with percentage of nuclear area
occupied by AgNOR in cells of the airways and
leukocytes in peripheral blood (Fig. 3b).
AgNOR Expression in Relation to Kitchen Location
Forty six out of 78 biomass-users (59%) lacked
separate kitchen. They used to cook in a space
adjacent to the living room. These women had
more AgNOR dots per nucleus in parabasal and
intermediate cells (6.47 ± 2.13 vs. 5.24 ± 2.18, p =
0.015), basal cells (7.15 ± 2.13 vs.5.98 ± 2.05, p =
0.017) and airway neutrophils (3.69 ± 1.17 vs.
3.10 ± 1.03, p = 0.024) than that of 32 biomass
users who possessed separate kitchen, and the
changes were statistically significant. Moreover,
c
d
b
a
Figure 2 Peripheral blood smears showing AgNOR dots in nuclei of (a and b) lymphocytes and (c and d)
neutrophils. Note increase in the size of AgNOR dots in (b) lymphocytes and (d) neutrophils of biomass users
relative to that of LPG users (a and c, respectively) (AgNOR staining, ×1,000; inset × 2,400(a,b), 1,700 (c,d)
Volume 33, Number 1/February 2011
AgNOR Expression in Biomass S
moke Exposure
56
women lacking separate kitchen had larger
AgNOR dots in parabasal and intermediate cells
(0.84 ± 0.22 vs. 0.80 ± 0.19 μm
2
, p = 0.406), basal
cells (0.85 ± 0.51 vs. 0.71 ± 0.42 μm
2
, p = 0.204),
airway neutrophils (1.84 ± 1.23 vs. 1.35 ± 1.09
μm
2
, p = 0.074), blood lymphocytes (0.99 ± 0.24
vs. 0.93 ± 0.32 μm
2
, p = 0.347) and blood
neutrophils (0.87 ± 0.27 vs. 0.76 ± 0.33 μm
2
, p =
0.110). However, the changes were not
statistically significant.
Association between Particulate Air Pollution in
Indoor Air and AgNOR Expression
Controlling education, family income and
kitchen location as possible confounders,
multivariate logistic regression analysis showed
a positive association between AgNOR
parameters and levels of PM
10
and PM
2.5
,
especially the latter, in indoor air (Table III).
Discussion
This study showed that chronic inhalation of
biomass smoke is associated with significant
increment in the number and area of AgNOR
dots in nuclei of airways cells and blood
leukocytes. AgNOR expression is not specific for
a single protein. Instead, it indicates the
expression of several silver-affinity proteins
belonging to ribosome assembly machinery
16
.
During interphase, nucleolin and
nucleophosmin are the two most important
AgNOR proteins
18
. Nucleolin is a specific
d
c
a
b
Table II Changes in AgNOR parameters in sputum and blood cells of biomass and LPG using women
Number of AgNOR
dots/nucleus
_______________________
Size of individual
AgNOR dot (μm
2
)
________________________
AgNOR- occupied
nuclear area (%)
________________________
Cell types
LPG user
Biomass user
LPG user
Biomass user
LPG user
Biomass user
Parabasal and intermediate
airway epithelial cells
3.05 ± 1.42
5.98 ± 2.06*
0.58 ± 0.17
0.82 ± 0.16*
1.82 ± 0.15
5.1
8 ± 1.49*
Airway basal epithelial cells
3.33 ± 1.04
6.67 ± 2.00*
0.27 ± 0.16
0.79 ± 0.41
1.48 ± 0.46
4.74 ± 1.08*
Airway neutrophil
1.41 ± 0.32
3.44 ± 1.01*
0.72 ± 0.35
1.65 ± 1.04*
1.57 ± 0.43
6.38 ± 1.01*
Peripheral blood lymphocyte
1.42 ± 0.40
1.52
± 0.33
0.42 ± 0.28
0.96 ± 0.24*
1.14 ± 0.43
2.08 ± 0.94*
Peripheral blood neutrophil
1.55 ± 0.39
1.67 ± 0.38
0.38 ± 0.11
0.82 ± 0.29*
0.95 ± 0.22
1.97 ± 0.86*
* p<0.05 compared with LPG-using control in Student t-test.
Mondal et al
Analytical and Quantitative Cytology and Histology®
initiator of rRNA transcription, and AgNOR
expression is considered an indicator of the rate
of ribosomes biogenesis
22
. Therefore, significant
increase in the number and size of AgNOR in
airway epithelial cells, airway neutrophils, and
peripheral blood leukocytes of biomass users of
this study suggests up-regulation of ribosome
biosynthesis in these cells. The present findings,
coupled with our earlier report of elevated
AgNOR expression in terminally differentiated
epithelial cells in buccal mucosa of biomass-
using women
23
, indicate higher rate of ribosome
biogenesis in cells present in the oropharynx, the
airways as well as in peripheral blood in
association with chronic exposure to biomass
smoke. It became also apparent that the
stimulatory effect of biomass smoke on AgNOR
expression is operative even in cells that are not
present at the direct route of exposure
(peripheral blood leukocytes). Moreover, the
effect was discernible in proliferating cells (basal
epithelial cells), terminally differentiated non-
proliferating cells (parabasal, intermediate and
squamous epithelial cells and neutrophils) as
Figure 3
Progressive
rise in the
(a) number of AgNOR dots per
nucleus and (b) percentage of
NOR-occupied nuclear area in
airway cells and blood
leukocytes with increasing
years of cooking with biomass
fuel. A, 5–9 years; B, 10–14
years; C, 15–19 years; and D,
20–24 years of cooking with
biomass. Vertical bars
represent standard deviation of
mean. *p<0.01 compared with
corresponding cells in group A
in Student t-test.
Table III Multivariate logistic regression analysis of association between AgNOR parameters with PM
10
and PM
2.5
levels in indoor air controlling potential confounders
Cell types and AgNOR parameters
With PM
10
______________________
With PM
2.5
_________________________
Odds ratio
95%
CI
Odds ratio
95%
CI
Airway epithelial cells
AgNOR dots per nucleus
1.25
1.03
1.52
1.35
1.12
1.65
Area of individual AgNOR dot
1.24
1.02
1.47
1.44
1.18
1.
76
Airway neutrophils
AgNOR dots per nucleus
1.33
1.12
1.56
1.68
1.35
1.96
Size of individual AgNOR dot
1.37
1.15
1.63
1.64
1.25
2.13
Peripheral blood lymphocytes
Size of individual AgNOR dot
1.29
1.06
1.57
2.50
1.25
3.66
Peripheral blood neutrophils
Size of individual AgNOR dot
1.24
1.04
1.64
1.89
1.66
2.23
All the associations were positive and statistically significant
Volume 33, Number 1/February 2011
AgNOR Expression in Biomass Smoke Exposure
58
well as in cells in G
0
phase of cell cycle
(lymphocytes).
Progressive increase in AgNOR expression
was found in association with years of cooking
with biomass fuels. Hence it is tempting to
conclude that the rise in AgNOR expression
among biomass-using women was due to the
cumulative effects of smoke exposures.
However, age of the participants and years of
cooking with either biomass or LPG were highly
correlated (r = 0.946 for biomass and 0.957 for
LPG; p<0.0001). Moreover, years of cooking
with LPG also had positive association with
AgNOR expression (data not shown). It is
reasonable to assume therefore that age itself is
an important determinant of AgNOR
expression, and progressive rise in AgNOR
expression in association with increasing
cooking years could be attributed to the
combined effect of age and cumulative smoke
exposure.
Lymphocytes in peripheral blood are
quiescent and contain one or two compact
AgNOR dots
24
while blood neutrophils have
inconspicuous irregular AgNOR. Our findings
are in general agreement with this. However,
the airway neutrophils illustrated significantly
higher AgNOR expression than that of blood
neutrophils in both LPG and biomass users. It is
well known that airway neutrophils are more
active than their circulating counterparts
25
.
Therefore, it seems that AgNOR expression has
an intimate association with cellular activity.
The reason for elevated AgNOR number and
size among biomass users is only speculative.
The participants of this study were all never-
smokers and non-chewers of tobacco and
exposure to environmental tobacco smoke was
similar among biomass and LPG users.
Therefore, the changes in AgNOR parameters in
biomass using women cannot be explained by
tobacco smoking or chewing habit which is a
much investigated cause of AgNOR
upregulation
26-29
. The villages where the
participants resided were far from the busy road
traffic and there were no air-polluting industries
within 5 km radius. Thus, ambient air pollution
levels in the study areas seemed negligible.
Moreover, biomass 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 biomass users and this could
explain to a large extent the observed increase in
AgNOR parameters. Biomass smoke contains a
host of mutagens and carcinogens including
benzo(a)pyrene, 1,3-butadiene and benzene
which are adsorbed on the surface of particulate
matters
6,30
. Since PM
10
and PM
2.5
levels were
59
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Analytical and Quantitative Cytology and Histology®
significantly elevated in indoor air of biomass-
using households, biomass-using women
appeared to be more exposed to these
pollutants. Intrapulmonary instillation of
benzo(a)pyrene in rats resulted in increase in
AgNOR dots in bronchial epithelial cells and the
number of dots per cell positively correlated
with the dose of the carcinogen
31
. Similarly, long
term exposure to sulfur dioxide, another air
pollutant, in rat increases the mean number of
AgNOR dots in epithelial cells
32
. Therefore,
upregulation of AgNOR expression can be
attributed in part to benzo(a)pyrene and oxides
of sulfur present in biomass smoke while other
pollutants present in smoke could also have
played a role.
Upregulation of AgNOR and rDNA
transcriptional activity has been linked to cell
proliferation
33-35
. Because of this, AgNOR
staining method has wide application in tumor
histopathology for evaluation of proliferating
activity and prognosis of malignant
neoplasms
36
. In the line of these reports,
elevated AgNOR expression in exfoliated basal
epithelial cells suggest cellular proliferation in
the airways of biomass users. However, increase
in AgNOR expression in airway neutrophils and
peripheral blood leukocytes among biomass
users is difficult to explain. Upregulation of
ribosome biogenesis in the airway neutrophils
probably indicates their altered metabolic
activities. Rise in the number and area of
AgNOR dots in peripheral blood leukocytes can
be mediated by ultrafine particles (particulate
matter with an aerodynamic diameter of less
than 0.1 micron) which are abundant in
biomass smoke and can readily cross alveolar-
capillary barrier following inhalation
37
causing
systemic alterations via oxidative stress
38
.
In essence, the study has shown that chronic
exposure to high level of IAP while cooking
with biomass fuel induces rise in the number of
AgNOR dots per nucleus as well as their size in
epithelial cells and neutrophils in the airways,
and leukocytes in peripheral blood, suggesting
upregulation of ribosome biogenesis in these
cells. This simple, cost-effective yet sensitive
cytochemical method can be utilized for
screening of cellular alterations with regard to
ribosome biogenesis in large population-based
studies in the developing nations where biomass
is still extensively used by the rural people. The
AgNOR technique can also be exploited to
identify high risk groups for the prevention of
cancer, because changes in AgNOR
characteristics help to differentiate hyperplastic,
pre-malignant, and malignant lesions
39-42
. Such
studies have immense public health importance
because lung cancer is the fifth leading cancer
type among predominantly never-smoking
women of eastern India where the present
investigation has been conducted
43
.
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... Longhin et al. 2013a;Mitkus et al. 2013;Mondal et al. 2011;Torres-Ramos et al. 2011). ...
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This study investigated the effect of indoor air pollution from biomass-fuel use on the expression of argyrophilic nucleolar organizer regions (AgNORs), an indicator of ribosome biosynthesis, in epithelial cells of oral mucosa. AgNORs were evaluated using cytochemical staining in 62 nonsmoking indian women (median age, 34 years), who cooked exclusively with biomass, and 55 age-matched women, who were from a similar neighborhood and cooked with relatively clean liquefied petroleum gas (LPG). Concentrations of particulate pollutants in indoor air were measured using a real-time aerosol monitor. Compared to the LPG-using controls, biomass-fuel users showed a remarkably increased number of AgNOR dots per nucleus (6.08 +/-2.26 vs 3.16 +/-0.86, p < 0.001), AgNOR size (0.85 +/-0.19 vs 0.53 +/-0.15 mum2, p < 0.001), and percentage of AgNOR-occupied nuclear area (4.88 +/-1.49 vs 1.75 +/-0.13%, p < 0.001). Biomass-using households had 2 to 4 times more particulate pollutants than that of LPG-using households. The changes in AgNOR expression were positively associated with PM10 and PM2.5 levels in indoor air after controlling for potential confounders such as age, kitchen location, and family income. Thus, biomass smoke appears to be a risk factor for abnormal cell growth via upregulation of ribosome biogenesis.
Article
Using a silver staining technique, nucleolar organizer region-associated proteins (Ag-NOR) were studied in paraffin sections of five specimens of normal bronchial epithelium, eight of atypical squamous metaplasia, five of carcinoma in situ, and seven of microinvasive squamous cell carcinoma. The mean number of Ag-NOR in the nucleus were normal epithelium 1.2 ± 0.1 (mean ± SD), atypical squamous metaplasia (borderline lesion) 2.2 ± 0.5, carcinoma in situ 3.8 ± 0.6, and microinvasive squamous cell carcinoma 4.8 ± 1.1. There was a highly significant difference between the Ag-NOR numbers in the atypical squamous metaplasia and those in the carcinoma in situ (P < 0.01). The Ag-NOR staining is a useful technique for the differential diagnosis of difficult borderline lesions in the bronchial epithelium.
Article
Nucleolar organizer regions (NORs), which are loops of DNA containing ribosomal RNA genes, have been shown to correlate with cell proliferation and malignant transformation. The diagnostic value of silver staining NORs (AgNORs) in preneoplastic epithelia and nasopharyngeal carcinoma (NPC) by the quantitative assessment of AgNORs proteins is evaluated. Silver-staining of NORs was applied to 70 paraffin sections of NPC biopsy specimens, including 29 samples of the adjacent normal glandular epithelia (GE), 19 hyperplastic/dysplastic columnar epithelia (CE) in the adjacent mucosa, 10 hyperplastic/dysplastic squamous epithelia (SE) in the adjacent mucosa, 54 differentiated nonkeratinizing carcinoma (DC), and 16 undifferentiated carcinoma (UC). The morphometric features of AgNORs in preneoplastic epithelia and cancer cells were analyzed by image cytometric analysis and then compared. The AgNOR count, mean AgNOR area, and AgNOR area–count ratio increased significantly from GE to CE to DC or UC. The mean nuclear area, AgNOR area, and AgNOR area–count ratio increased significantly from GE to SE to UC, whereas overlapping AgNOR values were observed between SE and DC. UC had significantly higher values than DC in mean nuclear area, AgNOR area, and AgNOR area–count ratio. The morphometric analysis of AgNORs reflected cell proliferative activity and histologic differentiation of tumor rather than malignant transformation in different nasopharyngeal epithelia and NPC. AgNOR area and AgNOR area–count ratio are the most valuable features for differential diagnosis of normal, preneoplastic, and cancer cells. © 2003 Wiley Periodicals, Inc. Head Neck 25:000–000, 2003
Article
In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin-fixed and paraffin-embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=−0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=−0.99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6±5.6 SD and 95.5±2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions. Copyright © 2000 John Wiley & Sons, Ltd.
Article
Using a silver staining technique, nucleolar organizer region-associated proteins (Ag-NORs) have been studied in paraffin sections of 90 non-Hodgkin's lymphomas, five palatine tonsils and five ‘reactive’ lymph nodes. The method was readily applicable to these preparations and the Ag-NORs were enumerated with ease. A significant difference was found between the numbers of Ag-NORs in the nuclei of low-grade lymphomas (from a mean of 1 to 1·5 per nucleus) and those of high-grade lymphomas (a mean of 4·4 to 6·8 per nucleus). The Ag-NOR regions were often observed in nuclei in areas where nucleoli themselves were not visible. It is suggested that this method, previously largely the province of the cytogeneticist, should find widespread applications in the field of tumour histopathology.
To compare the argyrophilic nucleolar organizer region (AgNOR) count of cells collected from the normal buccal mucosa of cigarette smokers, opium addicts and nonsmokers. Exfoliative cytologic smears of buccal mucosa from 25 smokers, 25 addicts and 25 nonsmokers were stained for AgNORs according to the Ploton's method. The AgNOR count was performed on 100 cells. These AgNOR counts were compared and analyzed using the SPSS 13 program (SPSS, Inc., Chicago, Illinois, U.S.A.) and one-way ANOVA test. Statistically, the highest mean AgNOR count (mAgNOR) was in cells from opium addicts (9.21 +/- 2.95) and the lowest in cells from nonsmokers (4.35 +/- 1.62) (p < 0.0001). For smokers, this value was in midrange (5.68 +/- 2.17). The percentage of cells with 6 or more AgNORs (pAgNOR > or =6) was the best discriminator among the different groups (p < 0.0001). Cigarette smoking and particularly opium abuse increases the rate of cellular proliferation in cells of normal buccal mucosa.
Article
A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.