EFFECTS OF PESTICIDE EXPOSURE AND TOBACCO

AMONG THE TEA GARDEN WORKERS OF CACHAR

DISTRICT, ASSAM

Presented by:- Project Guide:-

Soma Choudhury Dr.Prasenjit Roy.
T.D.C. 6th Semester
Department of
Biotechnology,
G.C.College, Silchar.
 Tea the truly ubiquitous common man’s drink in
India was introduced by the British to this
country from the neighbouring country China.
 Indian teas are appreciated around the world as
health drink for their unique flavor, aroma and
medicinal properties. Of the total tea produced
in India, a major portion is exported to countries
like United Kingdom, USA, Japan and
Germany.
 In India, Assam is the highest tea producing
state and a large number of workers are
employed in the various units of the tea
production industry. Tea is an important agro-
industry of Assam, which contributes
immensely to the state’s economy.
 Current trend of over- reliance on the use of
synthetic pesticides in tea crop protection
programs has resulted in disturbance to the
environment, pest resurgence, residue
problem in tea leaves and lethal effects on
non-target organisms including human beings.
 In other words, the chemical pesticides
represent a potential source of occupational
risk to humans.
 The tea garden workers are primarily exposed
to the pesticides through inhalation. However,
exposure also takes place through dermal
absorption or oral ingestion.
 Furthermore, in many cases norms of
pesticide handling, use of protective
equipments and the practice of washing hands
before eating and after handling the pesticides
is completely ignored. This happens mainly
because the workers lack knowledge about
the health hazards associated with the
application of these chemicals.
 Population biomonitoring is becoming an extremely
powerful approach to determine the effect of
environmental mutagens or occupation on human
populations.
 The tea garden workers consume areca nut with
“sadagura”. They also use other tobacco products
such as khaini, gutkha, pan masala and zarda. These
tea garden workers are barely aware of the impact of
tobacco usage on their health and therefore continue
to use it throughout their lifetime.
 The buccal micronucleus cytome(BMCyt) assay is a
non-invasive method for studying DNA damage,
chromosomal instability, cell death and the
regenerative potential of buccal mucosal tissue, which
is widely used in biomonitoring studies.
 Micronucleus (MN) assay has been used for the
evaluation of genotoxic effects in buccal epithelial
cells produced by carcinogenic substances (to which
human populations are exposed).
 It has been found that there is a significant increase in
the buccal cell micronuclei in human population
exposed to pesticides and tobacco.
 IMPACTS OF TOBACCO ON
HUMAN HEALTH
• Tobacco use is a leading cause of cancers and
of death from cancer.
• Nicotine is just one of over 4,000 chemicals
that can be found in tobacco and 19 of these
other chemicals in tobacco are known to be
carcinogenic.
• Nicotine is a psychoactive drug and highly
addictive in nature which makes it very
difficult to quit using tobacco products once
it has become a regular habit. Nicotine causes
release of dopamine(feel good hormone) in
the brain that control pleasure and
motivation.
• The main carcinogens in smokeless tobacco
are tobacco-specific nitrosamines(TSNAs),
arsenic, benzopyrene, nickel and cadmium.
• Although nicotine from smokeless tobacco is
absorbed more slowly than from cigarettes, 3 to
4 times more nicotine is absorbed from
smokeless tobacco than from cigarettes.
Nicotine from smokeless tobacco remains
longer in the bloodstream.
• According to ICAR, oral cancer is 11th most
common cancer and is responsible for 2,00,000
deaths every year worldwide.
• Smokeless tobacco uses have increased risk of
cardiovascular diseases.
• Those who use smokeless tobacco have an
increased risk of : developing gum diseases and
gum recession( pulling away of gum tissue
from the teeth); leukoplakia ( whitish patches
inside mouth that can become cancerous);
abrasion (wearing down) of teeth; staining of
teeth ; tooth decay; tooth loss and oral cancer.
BUCCAL MICRONUCLEUS CYTOME (BMCyt)
ASSAY
 The BMCyt assay is a minimally invasive method widely used in
molecular biological studies for measuring biomarkers of DNA damage
(micronuclei and/or nuclear buds), cytokinetic defects (binucleated cells)
and proliferative potential (basal cell frequency) and cell death (Thomas et
al., 2009).
 In molecular epidemiological studies, the BMCyt assay is significantly
used for investigating the impacts of environmental genotoxic agents,
nutrition, lifestyle factors, genotoxic exposure and genotype on DNA
damage, chromosomal aberrations and cell death (Thomas et al., 2009).
 Micronuclei are chromosomal fragments or the whole chromosomes that
are not included into the daughter nuclei during cell division and are
incorporated as a much smaller nucleus. The formation of micronucleus is
therefore induced by substances that cause breakage of chromosomes
(clastogens) as well as by agents which affect the spindle apparatus
(aneugens) (Ghosh et al., 2008).
 The micronucleus analysis in epithelial cells is relevant because about
90% of cancers are of epithelial origin (Rosin and Gilbert, 1990) and is
a reliable indicator for genomic instability.
 Exfoliated epithelial cells can be easily collected from mouth, nose by
non-invasive procedures and so are widely used as a tool for
biomonitoring genotoxic effects on population exposed to genotoxic
agents (Araújo et al., 2010).
 The aim of the present work is to observe micronuclei and
other nuclear abnormalities in the buccal epithelial cells of tea
garden workers.

Employing cytogenetic bio-monitoring study buccal epithelial

cells were analyzed in 10 male and 10 female tea garden workers
and compared to 20 tobacco non-chewers, unexposed to
pesticide, who are not tea garden workers.

The proposed work is done by using Buccal Micronucleus

Cytome (BMCyt) assay as described by Thomas et al., 2009 with
little modifications.
STUDY AREA AND SAMPLE POPULATION:.
For the present study, we have taken samples from the Tea garden workers of
Cachar district, Assam.

The samples are collected from 10 male and 10 female tea garden workers
and compared to 20 non-chewers, unexposed to pesticide, who are not tea
garden workers. A structured questionnaire based survey was also conducted.

TEST CHEMICALS
0.9% NaCl solution for buccal cell washing.

Methanol and acetic acid(3:1) for fixation.

Leishman stain for staining.

Distilled water for various dilution.

 0.9% NaCl was freshly prepared on the day of sample collection.

Taken into eppendorf tubes.

Cotton buds was used for sample collection.

Mouth of the individual was rinsed with clean water(repeat if mouth is still dry).

The cheek surface was swabbed for about 20 times in circular fashion to collect the
buccal cells.

The cells thus obtained were collected in the eppendorf tubes containing the prepared
NaCl solution.

The NaCl solutions inside the tubes were checked for turbidity indicating the presence
of sufficient cells.
 Centrifuged for 10 minutes at 2000 rpm.

Supernatants were discarded and the centrifugation was repeated for another
10 minutes.

Supernatants were discarded again and then centrifuged for 5 minutes at

1000 rpm.

The supernatant was discarded and the pellet was agitated with a syringe
for 3-4 minutes.

Smears were prepared on slides.

Dried and fixed in methanol and acetic acid (3:1) for 10 minutes.

Stained in Leishman stain for 10 minutes.

Thoroughly washed in distilled water (at least two times) , dried and studied

Figure: Simple presentation of the procedure used in the present study.

1000 clearly defined cells with distinct staining were selected manually in a
random fashion.

The slide is moved in an orderly manner from left to right and then down and
across in a stepwise manner on the stage in order to avoid measuring and
counting the same slide twice.

Micronuclear, binuclear and cells with nuclear bud were reported and noted.

The various types of cells thus observed under the microscope includes:

Normal Differentiated Cell

These cells have a uniformly stained nucleus, which is oval or round in shape.
They are distinguished from basal cells by their larger size and by a smaller
nucleus-to-cytoplasm ratio.
Cells with Micronuclei (MNi)
Cells with micronuclei are characterized by
the presence of both a main nucleus and one
or more smaller nuclear structures called
micronuclei (MNi). The micronuclei are
round or oval in shape and their diameter
should range between 1/3 and 1/16 of the
main nucleus.
 Binucleated Cells (BN)
Binucleated cells are cells containing two
main nuclei instead of one. The nuclei are
usually very close and may touch each other
and usually have the same morphology as
that observed in normal cells.
 Cells with Nuclear Bud (NBUD)
Cells with nuclear buds contain nuclei with
an apparent sharp constriction at one end of Figure:-Diagrammatic representation
of the process of formation of the
the nucleus suggestive of a budding process.
various cell types scored in BMCyt
assay.
 STATISTICAL ANALYSIS
Student’s t-test is used to determine the significance of the cellular
parameters using SPSS Software.

EXPERIMENTAL FINDINGS
STUDY DETAILS
Buccal Micronucleus Cytome Assay (Thomas et al., 2009) was used for
biomarker evaluation to assess the extent of cytological damage in 10
female and10 male tea garden workers and compared with control
individuals. 20 participants irrespective of age, gender and profession
were included in the study. Individuals were divided into two groups on
the basis of exposure. Group I participants are control and Group II
individuals are tea garden workers (Table 1).

Table 1: Characteristics of the study groups

Total Subjects
Groups Description Male Female
(n)
Group I Control 12 8 20
Group II Exposed 10 10 20
 A N A LY S E D D ATA
Micronucleus (MN)
Table 2 indicates the MN frequency for the studied group. The percentage of
MN cells for Group II was 5.05 when compared to Group I 0.30 and found to
be statistically significant when compared with control (P<0.001).
Table 2:Incidence of micronucleated cells in buccal epithelial cells of two groups
TOTAL % OF MICRONUCLEATED
STUDY GROUP SUBJECTS(n) CELLS(MEAN + SE) P VALUE

GROUP 1 20 0.30 + 0.12 -

GROUP 2 20 5.05 + 0.57 P<0.001

Figure 1: Histogram showing incidence of micronucleated cells in buccal epithelial cells of

two groups. Values are significantly different from control: *** P < 0.001.
 Nuclear Bud ( N B U D )
Table 3 describes the incidence of cells with NBUD among different groups. A
statistically significant increase in incidence of cells with NBUD was observed
when compared between Group II and Group I (P<0.001), indicating
genotoxic effects of chemicals in the exposed individuals.
Table 3: Incidence of cells with nuclear buds in buccal epithelial cells of two
groups.
TOTAL % OF MICRONUCLEATED
STUDY GROUP SUBJECTS(n) CELLS(MEAN + SE) P VALUE

GROUP 1 20 0.15 + 0.08 -

GROUP 2 20 3.36 + 0.42 P<0.001

Figure 2:Histogram showing incidence of cells with NBUD in buccal epithelial cells of
two groups. Values are significantly different from control: *** P < 0.001.
 Binucleated Cells( B N )
Table 4 describes the incidence of Binucleated cells among different groups.
Group II (4.75 ± 0.52) show statistically significant higher incidence of BN when
compared to control Group I (0.40 ± 0.19).
Table 4: Incidence of binucleated cells in buccal epithelial cells of two groups.

TOTAL % OF MICRONUCLEATED
STUDY GROUP SUBJECTS(n) CELLS(MEAN + SE) P VALUE

GROUP 1 20 0.40 + 0.19 -

GROUP 2 20 4.75 + 0.52 P<0.001

Figure 3:Histogram showing incidence of cells with binucleated cells in buccal epithelial
cells of two groups. Values are significantly different from control: *** P < 0.001.
 MICROSCOPIC OBSERVATION

Normal cell Micronucleated cell

Binucleated cell Cell with Nuclear Bud

 The study conducted by Department of Operative Dentistry, Dental College
and Hospital, Ahmedabad indicated that a significantly higher number of quid-
chewers suffered from bleeding gums, ulcers, etc in the oral cavity than non-
chewers. The clinical examination using the oral hygiene score indicated that
the oral hygiene status of quid chewers was deteriorated.
 As per study conducted by Department of Radiotherapy Chandigarh records of
about 100 consecutive cases of carcinoma of the buccal mucosa were treated in
their institute in a span of 2 years .
 According to the Institute of Life Science, Bhubaneswar, oral squamous cell
carcinoma(OSCE)is the sixth common cancer in the world.
 In Northeast India,the incidence of tobacco related oral cancer is reported to be
about 33%.(Kundu Sharbadeb et.al.,2017)
 According to the study conducted by Department of Life Science(Assam,
University, Silchar) “sadagura” a unique tobacco is harmful in inducing
genotoxicity and dangerous when taken with betel nut(Giri.et.al.,2008).
 Hence, study of the effect of tobacco quid chewers on buccal cell is a matter of
interest since tumour growth depends on a balance between cell death via
apoptosis and cell proliferation.
 According to the study conducted by International Agency for Research on
Cancer(IARC), 50% of the pesticide found to possess carcinogenic potential.
 The female workers are involved in collection of young tender leaves ideal for
making tea. The male workers are involved in the pesticide spraying activities.
 The pesticides used in the tea gardens belong to various chemical classes like
organophosphates and organochlorides, pyrethroids. Some of these pesticides have
been classified as “moderately hazardous” or by WHO (2005).
 Southern Assam is hot and extremely humid region which may cause pesticides to
remain in air for longer duration. The tea garden workers are primarily exposed to
the pesticides through inhalation. However, exposure also takes place through
dermal absorption or oral ingestion.
 Pesticides of different categories generate reactive oxygen species (ROS) which
can generate free radicals, resulting in lipid peroxidation (Kehrer, 1993;
Panemangalore et al., 1999) leading to damage of DNA molecule.
 All the tea garden workers chew tobacco along with betel nut. The buccal mucosal
cells have been considered to be more sensitive than lymphocytes to the induction
of the cytogenetic damage by genotoxicagents when the target tissue is epithelial
tissue.
 The buccal epithelial cells of the tea garden workers therefore get exposed to all
the mutagenic compnents of pesticides, areca nut and tobacco.
 The result of this study shows that levels of buccal micronucleus and other nuclear
parameters are higher in tea garden workers than in the (pesticide and tobacco unexposed)
control group.
 A statistically significant increase in incidence of micronucleated cells, binucleated cells,
cells with nuclear bud was observed in the tea garden workers when compared to the control
group.
 From the present study we can conclude that smokeless tobacco is not harmless. It induces
genotoxicity. Pesticide exposure and chewing areca nut along with smokeless tobacco use is
responsible for changes in cytome parameters in exfoliated buccal cells of the tea garden
workers.
 The results of this study provide novel information regarding correlation of genomic damage
with pesticide exposure and tobacco chewing habits.
 It is therefore necessary to educate the tea garden workers about the genotoxic effects of
tobacco and pesticide exposure in order to ensure that they take proper safety measures
while handling the harmful chemicals and get cautious about the repercussions of their
tobacco chewing habits.
 However, for establishing exfoliated cytology as a diagnostic tool for oral cancer, further
studies with a larger sample size needs to be carried out.
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