Effect of Organophosphorus on Biochemical Parameters on Agricultural Workers of Mango Orchards
Quazi S. Haque,
Male agricultural workers in the age group of 20-57 years employed in manual pesticide mixing, formulation and spraying at mango orchards at Malihabad and Mal, the mango belt in the vicinity of Lucknow were studied for occupational health risks. A control group belonging to similar socio-economic status and never involved either in agricultural operations or pesticide handling (reference group n = 50) was taken randomly from the same area for comparison purpose. Our findings revealed a high morbidity (76.6%) among the exposed population in contrast to (20%) observed in the reference group. The chief morbidity pertained to neuro symptoms and gastrointestinal problems as a result of occupational exposure to pesticides. Biochemical studies revealed that the acetyl-cholinesterase (AChE) activity in the blood was significantly lowered in the sprayers compared to the levels found in controls (p<0.01). Also, malondialdehyde level was significantly elevated among the sprayers (p<0.01) thereby indicating oxidative stress among the sprayers.
August 01, 2011; Accepted: November 15, 2011;
Published: January 10, 2012
India is basically an agricultural country. Nearly 70% of its population lives
in rural areas. The primary occupation of the rural population is agriculture.
A diverse group of agro-chemicals is indiscriminately sprayed by the farmers
for the pest control to save the crops. These agro-chemicals are popularly known
as pesticides which have been broadly classified into organo-phosphorus, organo-chlorine
pesticides, carbamates and pyrethroids etc. The most commonly sprayed pesticides
are Organophos Phorus (OP) insecticides which are very powerful neurotoxins
resulting in neurotoxicity on acute or chronic exposure. The accidental exposure
such as ingestion of OP pesticides in the case of suicidal attempts, a very
common practice in rural area of our country may cause pesticide poisoning which
may be fatal if unattended medically promptly resulting in mortality. The first
case of pesticide poisoning occurred in Kerala in 1958 where in over 100 people
died after consuming wheat flour contaminated with ethyl parathion known as
Folidol E 605. Another major case of pesticide poisoning occurred during Bhopal
gas leak (MIC) in 1984, killing thousands of people. The morbidity and the mortality
prevalence studies (Kesavachandran et al., 2006;
Rastogi et al., 2009) have shown that there
is an alarming rise in the rate of pesticide poisoning in rural India. OP pesticide
self poisoning is an important clinical problem in rural regions of the developing
world in including India that kills an estimated 200,000 people every year.
Exposure to even small amounts of an OP compound can be fatal; death is usually
caused by respiratory failure resulting from paralysis of diaphragm and intercostal
muscles. Deliberate self poisoning has reached epidemic proportions in rural
parts of India where the toxicity of available poisons and sparse medical facilities
ensures a high fatality rate of 20% is common. The occupational and environmental
exposure to pesticides is very high in the farming population and their families
including children due to para-occupational exposure resulting from storage
and dumping of these chemicals in their residential premises. It is, therefore
the rural community which is at a greater health risk due to consistent use
of the various pesticides. Earlier studies (Jevaratnam,
1990; Ferrer, 2003) have shown that the acute and
chronic exposure of pesticides through inhalation, in gestation and dermal absorption
may cause neurotoxicity (acute or delayed type) present a wide variety of symptoms
resulting from the inhibition of secretion and formation of acetyl-choline-esterase
enzyme by the pesticides directly. Due to non-formation of these enzymes there
is no breakdown of acetylcholine at the parasympathetic postganglionic nerve
endings resulting in excess accumulation of acetylcholine at the nerve endings
causing cholinergic syndrome which is reflected in the form of nicotinic and
muscaranic symptoms such as vagal inhibition, contraction of smooth muscles
of bronchioles, meiosis, increased tear secretion and salivary secretion , increased
GIT motility etc are some of the prominent clinical findings reported in the
literature (Von Ostem et al., 2004; Hernandez
et al., 2004). Some of the common earlier symptoms reported on acute
exposure to pesticide are headache, nausea, giddiness, gastric pain etc. among
the occupationally exposed agricultural workers (Wilson
et al., 2005; Fuzikawa et al., 2005).
As the root cause of pesticide poisoning is the significant decrease in the
formation of acetyl-choline-esterase enzyme and its activity is lowered by pesticide
it is mandatory to measure the activity and its level in the affected cases.
Bio-monitoring acetyl-choline-esterase is the fore-most important procedure
in clinical management of pesticide poisoning. Some of the investigators (Hernandez
et al., 2005) have recommended determination of acetyl-choline-esterase
activity in the blood (in isolated RBC). The enzyme formed in RBCs is
the True acetyl-choline-esterase while some studies (Anwar,
1997; Banerjee et al., 1999; Prakasam
et al., 2001) preferred to estimate pseudo-acetyl-cholinesterase
in plasma which is called as plasma butyryl choline-easterase (BuChE). It has
been found that both the formats of the enzyme are equally sensitive biomarkers
of pesticide poisoning. The present study was under taken to study the effect
of occupational organophosphate pesticide exposure on neuro-physiological and
gastrointestinal functions of pesticide sprayers engaged in mango plantation
at Malihabad and Mall areas in the vicinity of Lucknow. 150 male pesticide sprayers
in the age group of 20-57 years were randomly selected to the study.
MATERIALS AND METHODS
The proposed study selected randomly 150 OP pesticide exposed male rural agricultural
workers in the age group of 20 to 57 years having at least one years exposure
to OP pesticides as sprayers in the mango orchards in and around Mal and Malihabad
areas. A reference group (n = 50) belonging to similar socio-economic status
from the same areas were selected as controls having no history of exposure
to OP. The details of clothing worn and the protective devices, if used, are
noted on a pre designed survey proforma. These pesticide sprayers normally and
frequently sprayed insecticides to control pests of mangoes such as mealy bug,
mango hoppers and mango scale which cause a lot of damage during the flowering
and fruiting seasons. Some of the very common Op pesticides sprayed in mango
orchards are malathion, endosulfan, monochrotophus, methyl parathion, phosphomedon
and their mixture in different combinations. Spraying normally starts at the
end of December and continues until April/ May till the mango fruit ripened.
The sprayers are thus exposed to aerosols of pesticides during aerial spraying
as well as during manual mixing and loading. Pesticides application periods
last for 4 to 5 hours per week and the spraying operation covers an average
of 0.5 hectares area per day. The health examination was offered free of charge
to the pesticide applicators and sprayers working in the mango plantations in
Determination of cholinesterase activity: Acetyl-cholinesterase and
butyryl-cholinesterase activity in blood was estimated by the method of Elman
et al. (1961) as modified by Chambers and Chambers
(1989) by taking acetylcholine iodide as substrate and expressed as mmoles
hydrolyzed/h/l blood (I U-1). For assay, 0.025 mL of 25 fold diluted
blood was mixed with 0.015 mL of diluting buffer (Tris-HCl, 100 mM, pH 7.4)
containing 0.1 mM acetylcholine iodide. The reaction mixture was incubated for
15 min with constant shaking at 37°C and terminated with 0.5 mL mixture
of DTNB and SDS (0.04 and 44%, respectively, in diluting buffer). The absorbance
was read at 412 nm and converted to equivalent of mmoles hydrolyzed using molar
extinction coefficient of 13600 mole L-1 cm-1.
Estimation of glutathione: The level of GSH was estimated in the blood
by the method of Jallow et al. (1974). 0.5 mL
of blood was mixed with 1.5 mL of water, 2 mL of 10% TCA and centrifuged at
2000 rpm for 15 min. To the supernatant (1 mL), 4 mL 0.1 M phosphate buffer
(pH 7.4) and 0.1 mL of 0.4% DTNB in phosphate buffer was added and the color
was read at 412 nm.
Estimation of lipid peroxidation: Malondialdehyde (MDA) was estimated
in the blood by the method Stocks and Dormandy (1971).
0.5 mL of blood in phosphate buffer (pH 7.4, 100 mM) was incubated for 30 min
at 37°C and centrifuged. To the supernatant (3 mL) 1 mL 1% TBA was added
and kept in boiling water bath for 15 min. Contents were cooled in ice water
and centrifuged for 15 min at 2500 rpm. The absorbance was taken at 532 nm and
converted to equivalent of MDA (nmol/mL blood) using molar extinction coefficient
of 1.56x105 mol L-1 cm-1.
The personal and occupational details of the controls and exposed workers are
shown in Table 1. OP pesticides commonly sprayed by the workers
are given in Table 2a-c. The morbidity
profile in the exposed and control group is illustrated in Table
3 shows the prevalence of overall morbidity to be 76.6 vs. 20% in the exposed
workers and the reference group, respectively. The major morbidity observed
in the OP pesticide subjects was primarily neurological involving both central
and peripheral nervous systems. Moreover, in this study the result showed that
6.6% exposed workers exhibited signs and symptoms of peripheral neuropathy.
Out of 6.6% cases, half of them confirmed of affected by confirmed peripheral
neuropathy resulting from OP pesticide. Other morbidities such as respiratory,
ocular and Gastro-Intestinal Tract (GIT) were found to be quite prevalent in
the exposed group as exposure to pesticides involves routes of inhalation, ingestion
and dermal contact. The controls mainly suffered from GIT problems (Fig.
|| Sample size drawn from each area of the study with demographic
||Organophosphorus pesticides sprayed by the sprayers in Malihabad
|| OP pesticides sprayed in mango orchards in Mall area (Area-2)
|| OP pesticides sprayed in mango orchards in Rahimabad (Area-3)
The age wise distribution of morbidity pattern is shown in Table
4. The prevalence of different diseases showed increasing trend with respect
to age. The results indicated higher incidence of neurological diseases in >30
year age group in contrast to sample of <30 year age exposed to pesticide.
The subjects suffered more in >0 years age group (10.7 vs. 3.5%).
|| Prevalence of diseases in exposed and control groups
|| Age wise distribution of diseases in the exposed and control
||Prevalence of disease in control and exposed subjects, Controls
(N = 50) = 20.0%, Pesticide exposed workers (N = 150) = 76.6%
Other diseases such as gastro intestinal tract, cutaneous, ocular, musculo-skeletal
did not show significant differences between the two groups although the overall
prevalence of diseases was found to be significantly higher in senior age group
(86.1 vs. 69.4%; p<0.05) (Fig. 2).
||Age wise distribution of disease in the exposed and control
subjects, Control<30 years (N = 23) = 13.0%; Control>30 years (N =
27) = 25.9%, Exposed<30 years (N = 85) = 69.4%; Exposed>30 years (N
= 65) = 86.1%
There is compelling evidence from the results of the study that increased serum
level of lipid peroxides i.e., Malondialdehyde (MDA) was found in the pesticide
exposed population compared to the corresponding value estimated in the control
group. The increased values of MDA were maintained in relation to period of
exposure to the different mixtures of organophosphorus pesticide pesticides
sprayed in the mango orchard in the three areas of the study (Mall, Malihabad
and Rahimabad) from where the pesticide exposed population sample was drawn.
The significant increase in MDA activity was found to be associated with considerable
fall in the activity of glutathione (GSH) enzyme (p<0.001). The elevated
level of TBARS observed in this research could be due to decreased antioxidant
activity caused by exposure to pesticide mixtures which result in an increased
peroxidation of red cell membranes. In blood, normal erythrocyte function depends
on an intact cell membrane which is the target for many toxic OP pesticides.
It has been reported in pesticide exposed humans that these enzymes (MDA and
GSH) associated with the antioxidant defence mechanisms change under the influence
of pesticides. These enzymes efficiently scavenge toxic free radicals and are
partly responsible for protection against lipid peroxidation due to OP pesticide
exposure (Banerjee et al., 1999; Ranjbar
et al., 2002b). The correlation between TBARS and AChE activity found
in the present study is in agreement with the results obtained by other workers
(Akhgari et al., 2003; Singh
et al., 2007). The profile of the antioxidant enzymes (MDA, GSH)
in different stages of neurotoxicity, starting from sub-acute to chronic neurotoxicity
revealed decreased total antioxidant capacity (TAC and total thiol groups) and
increase in thiobarbituric reactive substances supporting earlier findings (Ranjbar
et al., 2002a; Handy et al., 2002;
Nasntiet et al., 2003). It has been strongly
suggested that in long-lasting exposure to OPIs, Reactive Oxygen Species (ROS)
simply consume and exhaust antioxidant agent present in the body which is the
reason why lower antioxidant capacity exists in chronic pesticide exposure while
in sub-chronic exposure, the body is capable with the persistent OPI-induced
reactive oxygen species in a longer period (Halliwell, 1994;
Stohs, 1995; Abdollahi et al.,
2004). The result of present study indicate that glutathione (GSH) linked
enzymes involved in cellular antioxidant defence system in human erythrocytes
were significantly affected (significant decrease in enzyme activity) following
exposure to mixtures of OP pesticides particularly in acute and chronic pesticide
The RBC AChE activities were significantly reduced in the pesticide exposed
workers compared to the control values and further data analysis showed that
there is a maximum fall in activities of these enzymes. Different studies have
recognized the invaluable role of AChE and BChE monitoring in rural agricultural
workers at high risk for exposure to OP pesticides (McCanley
et al., 2006). It was observed that the blood cholinesterase activity
needs at least a 15% decrease from an individuals normal level of plasma
or erythrocyte enzyme activity to be considered indicative of pesticide over
exposure. The acute and the chronic OP toxicity observed in this study was associated
with the significant inhibition of AChE enzymes in the normal function of the
nervous system which results in the accumulation of acetylcholine (ACh) in the
synaptic gap leading to disruption of the nervous Cholinesterase activity has
been one of the most important end points in assessing the exposure to OP pesticides.
Most occupational exposure to OP pesticides occurs from skin absorption, although
inhalation may be an important route of exposure during pesticide manufacture
and application. Skin absorption can occur when dermal contact is made during
handling and application of the insecticides, as most of the farm workers never
use any Personal Protective Equipment (PPE) but prefer to work with bare bodies
system (Domingues et al., 2010). RBC AChE regeneration
is restored only as new red blood cells are formed (Regeneration of red blood
cells takes placed at a rate of about b1% per day). Most OPs need activation
within the body to become anticholinergic compounds and there are differences
between individuals in their ability to both activate and detoxify Ops.
Measurement of biochemical parameters in the blood of applicators suggested that their exposure to pesticides may contribute to decrease in AChE activity and increase in MDA both surrogate measurements for adverse effects from pesticide exposure. The use of pesticides in mango production is an important input by farmers. However, pesticide use may also present health concern to those making the application. Result of the study show that pesticide applicators complained of gastrointestinal, dermal and respiratory problems. The study based on the finding suggests selective and efficient use of protective measure (use of personal protective equipments and to conduct more awareness programs for rural community.
The authors thank the UGC for financial assistance to the department under
the SAP and Ministry of Science and Technology for financial assistance under
the DST-FIST programme. Support from IITR for carrying out biochemical work
is gratefully acknowledged.
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