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Declaration I Herby declare that this submission is my own work and that, to the best of
my knowledge and belief, it contain no material previously published or written
by another person nor material which to a substantial extent has been
accepted for the award of any other degree of the university of other institute,
except where due acknowledgment has been made in the text.
Signature Name Date
TAHA Taha Abdallah T. El Shanty 19 August 2009
Copy right All rights reserved: No part of this work may be reproduced, stored in a
retrieval system, or transmitted, in any form or by any means, electronic,
mechanical, photocopying, recording or otherwise without the prior author's
permission obtained.
Dedication
To my late Father and Mother,
my sister Jameela who supported me along the time,
my brothers,
my wife,
my children,
my Islamic University and to all I love.
Taha El- Shanty
Acknowledgments
It is impossible to convey, in a couple of sentences, my gratitude to many
people for helping me to learn and who cooperation made this work possible.
All praises and glory are due to Allah for all the bounty and support granted to
me.
I would like to express my gratitude to my supervisor Prof. Dr. Maged
Yassin, Professor of Human Physiology, Faculty of Medicine, The Islamic
University of Gaza for his initiating, planning of this work and for his valuable
scientific advices. He stood with me step by step and he was very keen to
show me every thing right. He introduced me to the world of scientific
research in an international atmosphere with interesting and challenging
project. His never-ending optimism, energy and intelligence earned my great
respect.
I wish to give my warmest thanks for all the members of El-Dora and
El-Nasser Hospitals Laboratory team specially for Mr. Abd El-Hakem El-
Jadba and Mr. Jamal Deeb for their help and clinical assistance.
My thanks shoud be extended to Mr. Abd El- Rahman Hamad for his
help in data analysis.
I wish to thank the head of the Master Program, Assistant professor
Abboud El Kichaoui and I am grateful to all the staff members and the
colleagues of the Master Program of Medical Technology.
Finally, I am in deepest gratitude to all of those individuals for their boundless support.
Taha El- Shanty
Abstract Background: Organophosphorus pesticides (OP) are toxic substances
frequently used in the Gaza Strip to combat insects, rodents and plants pests
and other creatures that can pose problems for agriculture, public health,
homes, schools, buildings and communities. Children are the most vulnerable
victims for organophosphorus pesticides poisoning (OPP). Objective: To assess risk factors, diagnosed symptoms and biochemical
alterations associated with OPP among children in Gaza City, Gaza strip.
Materials and methods: In this cross sectional study, data were obtained
from questionnaire interview, and biochemical analysis of blood of 104 OP
poisoned children aged 1-12 years admitted to El-Dorah and El-Nasser
hospitals in Gaza City and 97 healthy individuals as a control group. Data
were analyzed statistically by using SPSS program. Results: The total response for the questionnaire interview was 46.2% (n =
48). Eating of poisoned biscuits, bread or meat were commonly contributed to
the poisoning cases among children 21 (43.8%). Large number of children
sponsors 31 (64.6%) reported the use pesticides in their houses. A total of 4
(8.3%) admitted the recent treatment of their children with head lice. Out of 18
children sponsors, 12 (66.7%) said that they spraying their gardens or farms
in the presence of their children and 13 (72.2%) of them reported that they did
not store pesticide bottles in safe place. The most common diagnosed
symptoms among children were pin point pupils 53 (51.0%), vomiting 46
(44.2%), drawsy 42 (40.4%), conscious 40 (38.5%) and convulsions 30
(28.8%). The mean serum cholinesterase in cases was significantly lower
than that in controls (2004.3±1181.9 v 6659.8±1199.2 u/l, % difference=
69.9%, P=0.000). Changes in the enzyme activity was significantly associated
with drawsy, conscious and pin point (p=0.001, 0.024 and 0.030,
respectively). There was also a statistically significant increase in glucose
level of the cases compared to the controls (135.9±44.0 v 69.5±13.1 mg/dl, %
difference=95.5, P=0.000). The average levels of serum Alanine
Aminotransferase(ALT), Aspartate Aminotransferase (AST) and Alkaline
Phosphatase (ALP) in the cases (16.6±6.9, 19.8±8.5 and 319.0±114.3 u/l,
respectively) were significantly higher than those in the controls (11.2±2.6,
13.6±4.2 and 245.2±70.1 u/l, respectively) with percentage differences of
48.2, 45.6 and 30.1, respectively and p=0.000. Urea and creatinine levels
were increased significantly in the cases compared to the controls (21.0±9.3 v
15.2±5.0 mg/dl, % difference=38.2 and 0.50±0.13 v 0.39±0.11 mg/dl, %
difference=28.2, respectively, P=0.000). Potassium and phosphorus were
decreased significantly in the cases than the controls (4.0±0.45 v 4.3±0.46
meq/l, % difference=7.0 and 4.1±0.79 v 4.7±1.2 mg/dl, % difference=12.8
p=0.000, respectively). There were also significant decreases in total protein,
albumin and globulin in the cases compared to the controls showing %
differences of 8.3, 2.8 and 21.8, respectively (5.5±0.80, 3.5±0.39 and
1.97±0.68 v 6.0±0.74, 3.6±0.57 and 2.52±0.72 g/dl, with p=0.000, 0.046 and
0.000, respectively). White blood cell and platelets counts were higher in the
cases (14.8±7.0 v 7.5±1.5 X103cell/µl, % difference=97.3 and 398.3±163.6 v
307.2±59.9 X103cell/µl, % difference=29.7 respectively, p=0.000).
Hemoglobin and hematocrit were significantly lower in the cases (10.8±1.1
mg/dl and 33.4±3.7% v 12.0±0.87 mg/dl and 37.1±2.4 %, % differences=10.0,
p=0.000). MCV and MCH were also found to be significantly lower in the
cases (73.16±8.33 fl and 23.61±2.76 pg V 84.13±4.32 fl and 27.25±1.53 pg,
% differences=13.0 and 13.4 respectively, p=0.000). Pearson's correlation
test showed negative significant correlation between cholinesterase and
glucose, ALT, AST or creatinine (r=0.321, 0.291, 0.210, 0.212 and p=0.001,
0.003, 0.032 and 0.030, respectively). On the other hand, positive significant
correlation between cholinesterase and phosphorus was achieved (r=0.234,
p=0.017).
Key words: Children, Gaza City, Organophosphorus pesticides poisoning
ص الرسالة باللغة العربيةلخستم
العضوية في مدينة غزة الفسفور تتسمم األطفال بمبيدا
مة مقد
العضوية هي مواد سامة تستخدم عادة في قطـاع غـزة للقضـاء علـى الفسفورمبيدات
الحشرات و القوارض و اآلفات الزراعية و مختلف الكائنات التي تسبب مشاكل للزراعـة و
كثـر األطفـال هـم األ أنزل والمدارس والمباني و المؤسسات حيث الصحة العامة و المنا
.العضوية ورالفسفعرضة للتسمم بمبيدات
هدف الدراسة
صاحبة مع التسـمم يوكيميائية المتحديد عوامل الخطر و تشخيص األعراض و التغيرات الب
. العضوية عند األطفال في مدينة غزة سفورالفبمبيدات
المواد والخطوات
ى كيميائية للدم علبيوباستخدام مقابلة شخصية و تحاليل فيها هذه الدراسة تم جمع البيانات
عام دخلوا مستشفيات الدرة و النصر بمدينة 12-1أعمارهم من أطفال تسمموا كانت 104
spssطفل سليم كعينة ضابطة وتم تحليل البيانات إحصائيا باستخدام برنامج 97و ,غزة
.
ج النتائ
لحم به أكلوا بسكويت أو خبز أو ومنهم من%) 46.2(طفل 48مقابلة الشخصية لل استجاب
) %64.6(طفـل 31عدد كبير من ذوي األطفـال واستخدم ,%)43.8(طفل 21ا سم كانو
. دخلوا المشفى نتيجة استخدام مبيدات للقمل%) 8.3(أربع حاالت ,المبيدات في منازلهم
طفـل 12ذوي ذكـر ,بالمبيـدات حـدائقهم أو مـزارعهم طفل كانوا يرشون 18ذوي
قالوا أنهـم %) 72.2( 13و ,جود أطفالهمفي ومنهم قالوا أنهم كانوا يرشون %) 66.7(
.األطفال ال يخزنون المبيدات في مكان آمن من
46ثـم التقيـؤ %) 51(طفـل 53أكثر األعراض بين األطفال كان زوغان في العـين
ثـم %) 38.5(طفـل 40 فقـدان الـوعي ثم%) 40.4(طفل 42ثم الدوخان ) 44.2(طفل
).28.85(طفل 30 تشنجات
ن متوسط مستوى أنزيم كولين استيريز في األطفال المصـابين بالتسـمم أظهرت النتائج أ
أو فقـدان للـوعي دوخة أو صاحب ذلكو% . 69.9كانت اقل من العينة الضابطة بنسبة
زوغان في العين و كان هناك ارتفاع في نسبة السكر و أنزيمات الكبد و ووظـائف الكلـى
ور وفـي البـروتين و األلبـومين و وكان هناك نقص في أمـالح البوتاسـيوم والفسـف
و كان هناك ارتفاع في نسبة كرات الدم البيضاء و الصفائح الدموية و نقـص ,الجلوبيولين
. في الهيموجلوبين و الهيماتوكريت مقارنة مع العينة الضابطة
Table of contents Contents
Page
Declaration………………………………………………………………….. II
Copy right………………………………………………………………….. III
Dedication…………………………………………………………………… IV
Acknowledgment……………………………………………………………. V
English abstract ...…………………………………………………………… VI
Arabic abstract ………………………………………………………………. VIII
Table of contents……………………………………………………………. X
List of tables ……………………………………………………………….. XIII
List of figures ……………………………………………………………… XIII
Chapter 1 Introduction Introduction………………………………………………………………….. 1
1.1. Overview ……………………………………………………………... 1
1.2. Objectives …………………………………………………………… 3
1.3. Significance …………………………………………………………… 3
1.4. Study area ……………………………………………………………… 4
Chapter 2 Literature Review 2.1. Definition of pesticide………………………………………………… 6
2.2. Organophosphorus pesticides ………………………………………… 6
2.3. Risk factors of organophosphorus pesticides exposure in children…… 7
2.4. Pathophysiology of organophosphorus pesticide poisoning …………… 10
2.5. Toxicity symptoms of organophosphorus pesticide poisoning ………… 12
2.6. Effect of organophosphorus pesticides on body organs…………… 15
2.6.1 Effect of organophosphorus pesticides on liver ……………………… 15
2.6.2 Effect of organophosphorus pesticides on kidney …………………… 17
2.6.3 Effect of organophosphorus pesticides on blood …………………. 18
Chapter 3 Materials and Methods 3.1 Study Design, Period of the study, Setting of the study, Study
Population, sample size and Sampling. ……………………………………
21
3.7 Ethical consideration and Samples collection and processing ………… 22
3.9 Questionnaire interview………………………………………………… 22
3.10 Biochemical analysis ………………………………………………… 23
3.10.1 Determination of cholinesterase activity …………………………… 23
3.10.2 Determination of serum glucose ……………………………………. 24
3.10.3 Determination of alanine aminotransferase (ALT) activity …………. 25
3.10.4 Determination of aspartate aminotransferase (AST) activity ……… 27
3.10.5 Determination of alkaline phosphatase (ALP) activity………………. 29
3.10.6 Determination of urea…………………………….………………… 30
3.10.7 Determination of creatinine ……...………………………………… 32
3.10.8 Determination of Total protein……………………………………. 34
3.10.9 Determination of Albumin ………………………………….……. 35
3.10.10 Determination of Globulin ………………………………………. 36
3.10.11 Determination of serum Electrolyte ……………………………… 36
3.10.12 Determination of serum phosphorus ……………………………… 36
3.11 Hematological analysis ……………………………………………… 38
3.12 Statistical analysis……………………………………………………… 38
Chapter 4 Results 4.1 General characteristics of the study population ………….……………. 39
4.2 Questionnaire interview ………………………………………………. 41
4.3 Diagnosed symptoms………………………………………………… 43
4.4 Biochemical analysis …………………………………………………… 43
4.4.1 Serum Cholinesterase………………………….……………………… 43
4.4.2 Serum glucose……………………………..………………………… 45
4.4.3 Liver function. ……….……………………………………………… 46
4.4.4 Protein profile ………………………………………………………… 46
4.4.4.1 Non protein nitrogen constituent…………………………………… 46
4.4.4.2 Protein nitrogen constituent………………………………………… 47
4.4.5 Serum electrolytes …………………………………………………… 47
4.4.6 Hematological parameters…………………………..……………… 48
4.4.7 Association between cholinesterase with some parameters…………. 50
Chapter 5 Discussion Discussion………………………………………………………………. 52
Chapter 6 Conclusion and Recommendation 6.1 Conclusion………………………………………………………………. 60
6.2 Recommendation……………………………………………………… 61
Chapter 7 References References………………………………………………………………. 62
Annexes……………………………………………………….... .......... 75
List of Tables Table 1: Causes of pesticides poisoning among children as reported by their
sponsors (n=48) …………………………………………………………………….
41
Table 2: Domestic use of pesticides, treatment of pets (especially cats/dogs)
with pesticides, recent pesticides treatment of children with head lice as
reported by children sponsors (n=48) …………………………………………
42
Table3: Garden or farm activities as reported by children sponsors (n=48) …… 42
Table 4: Diagnosed symptoms among poisoned children (n=104) ……………… 43
Table 5: Serum Cholinesterase enzyme activity of the cases and the controls … 43
Table 6: Mean differences of cholinesterase activities among the most common
diagnosed symptoms in patients (n=104) …………………………………………
45
Table 7: Serum glucose of the controls and the cases …………………………… 45
Table 8: Serum liver enzyme of the case study and the controls ………………… 46
Table 9: Serum kidney function of the case study and the controls ……………… 46
Table 10: Serum protein profile (Total protein, albumin and globulin) of the
cases and the controls…………………………………………………………………
47
Table 11: Serum electrolytes of the case study and the controls …............. 48
Table 12: White blood cells and blood platelets of the cases and the controls … 49
Table 13: Primary secondary blood indices of the cases and the controls ……… 50
Table 14: Pearson's correlation between serum cholinesterase and serum
glucose, phosphorus and total protein ………………………………………………
50
Table 15: Pearson's correlation between serum cholinesterase and serum AST,
ALT and ALP…………………………………………………………………………
51
Table 16: Pearson's correlation between serum cholinesterase and serum Urea
and Creatinine …………………………………………………………………………
51
List of Figures Figure 2.1:Pathophysiology of organophosphorus pesticide poisoning…… 10
Figure 4.1:Distribuion of the study population by gender ……………… 39
Figure 4.2: Distribution of the cases (n=104) by Gaza City hospitals 40
Figure 4.3: Mean age of the controls and the cases…………………… 40
Figure 4.4: cholinesterase activity among the most common
diagnosed symptoms in organophosphorus poisoned children and
in healthy controls in Gaza City …………………………………………
44
Chapter 1
Introduction
1.1 Overview
Pesticides are substances or a mixture of substances intended to control a
variety of pests such as insects, rodents, fungi, weeds microorganisms, and
other unwanted organisms. Pesticides are usually classified into insecticides,
fungicides and herbicides. Other categories include rodenticides, termiticides,
miticides, disinfectants and insect repellents (Keifer, 1997).
Many Organophosphorus pesticides (OP) in use today belong to the
phosphorothionate group which includes chlorpyrifos, parathion and
tebupirimphos; phosphorodithioate group which includes malathion,
disulfoton, azinphos-methyl, sulprofos and dimethoate and
phosphoroamidothiolates group which includes acephate and methamidophos
(Mileson et al. 1998).
Organophosphorus pesticides are toxic substances frequently used to
combat insects, rodents and plants pests and other creatures that can pose
problems for agriculture, public health, or homes, schools, buildings and
communities (World health organization (WHO), 1990).
Organophosphorus pesticides are biologically active compounds, used
often in pest combat without any medical control, remain an important source
of poisoning. Intoxication associated with OP exposure usually follows
absorption either through skin, by inhalation or by ingestion (Iorrizo et al.,
1996). Organophosphorus pesticides are extensively used in Gaza strip and
the developing countries with applying poor protective measures (Forget,
1991 and Yassin et al., 2002). Several cases of chronic toxicity or death have
been reported and proven among humans exposed to different types of OP in
the Gaza Strip and other developing countries (El-Sebae, 1993; Safi, 2000
and Eddleston et al., 2006).
Young children may be highly exposed to OP because of their normal
tendency to explore their environment orally, combined with their proximity to
potentially contaminated floors, surfaces and air. The breathing zone for an
adult is typically four to six feet above the floor. A child's breathing zone is
closer to the floor. Heavier chemicals and large respirable particulates settle
out within these lower zones. Air concentration of pesticides have been found
to be higher closer to the floor (Fensake et al., 1991). In addition, physiologic
characteristics of young children, such as high intake of food, water, and air
per unit of body weight, may also increase their exposure (National Research
council, 1993).
Farmers' children can be exposed to OP through consumption of
contaminated food, by household use of pesticides, as a result of drift from
nearby agriculture applications, by contaminated breast milk from their farm
worker mothers, by playing in the fields, and through pesticides tracked into
their homes by their parents or other household members working in fields
(Eskenazi et al.,1999). Organophosphorus pesticides poisoning (OPP) also
occurs as a result of deliberate exposure to OP (Eddleston, 2000, Eddleston,
Murry, 2001 and Karalliedde et al., 2001).
The biomarker indicating OPP is the enzyme cholinesterase. There are
two types of cholinesterase in humans: acetylcholinesterase (AChE) which is
identified in nervous system and blood and butyrylcholinesterase (BuChE)
which is present only in blood and named as serum cholinesterase.
Acetylcholinesterase and BuChE are inhibited irreversibly by
organophosphorus pesticides (Padella, 1995 and Wexler, 1998). The most
practical test for monitoring of humans exposed to OP is the test for serum
cholinesterase levels (Hillman, 1994 and Safi et al., 2005). Inhibition of brain
AChE at synapses by OPP results in accumulation of acetylcholine and over
activation of acetylcholine receptor at neuromuscular junction and in the
autonomic and central nervous system. This will manifested in convulsions
and even tremors leading in sever cases to death (Lotti, 2001).
Organophosphorus pesticides also have harmful effects on other body
organs including liver and kidney (Yassin, 2003). Children may have an
increased sensitivity to pesticides due to their small body mass and because
they differ physiologically from adults. For instance, in newborns, liver function
has not reached its full potential; thus concentrations of enzymes needed to
detoxify certain pesticides may be low (Keifer, 1997).
Although data on OPP in Gaza Strip are restricted to farmers (Yassin
et al., 2002 and Safi et al., 2005), no published data are available on OPP in
children. The present study will assess for the first time the OPP and the
associated risk factors among children in Gaza City, Gaza Strip. This will
expand our knowledge on OPP in children and will be helpful in determination
of poisoning severity which will be useful in the treatment strategy against
these highly toxic compounds.
1.2 Objectives
The overall aim of the present study is to define risk factors, diagnosed
symptoms and some biochemical alterations associated with OPP among
children in Gaza City, Gaza strip.
The specific objectives are: 1. To identify the risk factors of OPP in children.
2. To describe the diagnosed symptoms associated with OPP.
3. To measure serum cholinesterase as indicator for OPP and correlate it with
other biochemical parameters.
4. To determine glucose levels in OP poisoned children and compared them
with the controls.
5. To assess liver and kidney functions in the children poisoned with OP and
compared them with the controls.
6. To examine electrolyte and protein profiles in OP poisoned children and
compared them with the controls.
7. To measure CBC of poisoned and the control children.
1.3 Significance 1. This will be the first study to assess various aspect of OPP among children
in Gaza City.
2. It is important to aware the child's family from risk factors of OPP that will
be determined in this study.
3. The amount of serum cholinesterase reduction due to OPP will be used as
an indicator of poisoning severity. Knowing severity of poisoning could help us
to choose the suitable dose of atropine for treatment and to determine the
follow up period of treatment.
4. Testing correlation between serum cholinesterase and other biochemical
parameters will be useful in determination of the most affected parameters
during OPP.
1.4 Study area
The Gaza strip is an elongated area located in a semi-arid region. It is
bordered by Egypt from the South, the Negev Desert from the East, and the
Mediterranean Sea from the West. The total surface area of the Gaza Strip is
365 Km2 and its population is estimated to be 1.416.543 people. Gaza city is
located in the North of the Gaza Strip. The surface area of the Gaza city is
45 Km2 and its population is estimated to be 496.411 people. The number of
people aged 0-14 years in Gaza City totals 234,368 or 47.2% of the total
Gaza City population (Palestinian Bureau of Statistics, 2007). There are two
Governmental hospitals for children in Gaza city (El-Dorah in the East and El-
Nasser in the West). Excessive use of pesticides, inadequate disposal of
municipal solid waste and improper design of sewage network infrastructure
are the main environmental health problems in the Gaza Strip (Yassin et al.,
2002; Tubail et al., 2004 and Yassin et al., 2008). Agriculture is the backbone
of economy in the Gaza Strip. More than 250 metric tons of formulated
pesticides, mainly insecticides and fungicides, in addition to one thousand
metric tons of methyl bromide, are used annually in the Gaza Strip. Some of
these pesticides have been internationally suspended, banned or cancelled
because of their mutagenecity, teratogenecity or carcinogenicity (Safi et al.,
2005). Several cases of toxicity or death have been reported and proven
among humans exposed to different types of OP in the Gaza strip (Safi,
2000). This may be a result of the use and or misuse of OP pesticides.
Inadequate disposal of municipal solid waste and improper design of sewage
network infrastructure in Gaza City, constitute a suitable environment for
breading of pests including rodents. This forced people to combat such pests
by using different organophosphorus poisons at a large scale. Children are
the most vulnerable victims for such poisons because of their behavior and
playing activities (Tulve et al., 2002).
Chapter 2
Literature Review
2.1 Definition of pesticide
A pesticide is any substance or mixture of substances intended for preventing,
destroying, repelling, or mitigating any pest. Pests can be insects, mice and
other animals, unwanted plants (weeds), fungi, or microorganisms like
bacteria and viruses. Though often misunderstood to refer only to insecticides
(kill insects and other arthropods), the term pesticide also applies to
herbicides (kill weeds and other plants that grow where they are not wanted),
fungicides (kill fungi including blights, mildews, molds, and rusts),
Rodenticides (control mice and other rodents), and various other substances
used to control pests. Under United States law, a pesticide is also any
substance or mixture of substances intended for use as a plant regulator,
defoliant, or desiccant (Environmental Protection Agency, EPA, 2006).
2.2 Organophosphorus pesticides
Organophosphorus pesticides (OP) are highly toxic compounds containing
active phosphorus. They are classified into three groups: phosphorothionate
group, in which phosphorus is bound to three oxygens and one sulfur (the
double bond). Phosphorothionates include chlorpyrifos, parathion, and
tebupirimphos. Compounds in the phosphorodithioate group are like the
phosphorothionates but with one of the oxygens replaced by sulfur.
Phosphorodithioates include malathion, disulfoton, azinphos-methyl,
sulprofos, and dimethoate. The atoms bound to the Phosphorus of
phosphoroamidothiolates are nitrogen, sulfur, and two oxygens; the double
bond is to an oxygen. Examples of phosphoroamidothiolates are acephate
and methamidophos (Chambers, 1992).
Organophosphorus pesticides are used widely for agriculture, vector
control, and domestic purposes. Despite the apparent benefits of these uses,
OP are harmful to humans, particularly children. It has been reported an
estimated 1 to 5 million cases of pesticide poisonings occur every year,
resulting in 20,000 fatalities. Most of these poisoning take place in developing
countries, where safeguards typically are inadequate or lacking altogether
(Eddleston, 2000; Jeyaratnam, 1990; WHO, and 1990). Although the
incidence of severe acute organophosphorus pesticide poisoning is much less
in developed countries, many patients with acute low dose unintentional or
occupational exposures present to health facilities (Roberts et al., 2005 and
Lai et al., 2006).
Many household products are pesticides such as:
• Cockroach sprays and baits
• Insect repellents for personal use.
• Rat and other rodent poisons.
• Flea and tick sprays, powders, and pet collars.
• Kitchen, laundry, and bath disinfectants and sanitizers.
• Products that kill mold and mildew.
• Some lawn and garden products, such as weed killers.
• Some swimming pool chemicals.
2.3 Risk factors of organophosphorus pesticides exposure in children Exposure in the home depends on the frequency, duration and nature (i.e.,
dermal contact, hand to mouth behavior) of the child's interaction with
contaminated media such as house dust. Children may have higher exposure
to pesticides than other residents living in the same contaminated
environment, in part because young children spend more of their time indoors
at home (Silvers et al., 1994).
Children's behaviors also put them at high risk. They crawl on floors
and play on lawns-places where pesticide residues collect-and put objects
into their mouths. They consume more milk, applesauce, apple and orange
juice-foods that may have high pesticide loads-per pound of body weight than
adults. Children of farm workers are at especial risk for pesticide exposure.
Their parents may bring pesticide residues from the agricultural fields into the
home, and pesticides may drift from fields into areas where children play.
Although many people associate pesticides problems with agriculture, many
products used around the home and schools are also dangerous, insect
repellents for personal use, flea and tick sprays, powders, and pet collar,
products that kill mold and mildew, weed killers, and swimming pool
chemicals are examples (Roberts and Aaron, 2007).
Pesticides are often applied as aerosols, and sprayed in the vicinity of
children, inhalation exposure poses a common health threat to children. This
is especially true in the agricultural setting, in which the dwellings of farm
laborers are often adjacent to crops that are sprayed in this setting, children
who work or play in fields are sometimes sprayed accidentally or from
neighboring fields (Wilk, 1990).
Children eat more food relative to their body weights than adults do,
and because children consume, on average, more contaminated fruits, they
face higher risks than adults from pesticide residues (Natural Resources
Defense Council, 1989). Ingestion exposure may also occur via contaminated
beverages, drinking water, breast milk, hand to mouth contact, and pica
behavior or soil ingestion. Oral poisonings, occurred when the child drank
improperly stored liquid agents, contacted a contaminated container or object,
or ate insecticide granules (ZWeiner and Ginsburg, 1988).
Because children's playing habits involve contact with many surfaces,
dermal exposure by playing with surfaces or dirt contaminated with pesticides,
and exposed parents provide opportunities for dermal exposure via clothing
and skin (Fenske et al., 1990).
The exposure potential for children of agricultural families may be
higher than for other child populations because concentrated formulation of
pesticides are used in high volume near the home. Pesticides used during
work also may be introduced into the home inadvertently via various take-
home pathways. This type of exposure, often referred to as paraoccupational
exposure, has been well documented for a number of industrial chemicals
and was the subject of a report of the U.S. (National Institute for Occupational
Safety and Health ;NOISH, 1993). The result of a New York Stale survey
illustrated an extreme case of agricultural pesticides exposure: of 50 migrant
farm worker children, 36% had been sprayed in the fields, and 34% replied
that farm dwelling had been sprayed (Pollack and Landrigan 1990).
In West Bank, Saleh et al., (1995) pointed out that pesticide related
injury cases were found in 26% of the farmer interviewed. The injuries were
not restricted to the sprayers themselves, having also affected women and
children, because of their participation in spraying process. From all reported
cases, 50% were injuries caused by pesticides named karate, lannate,
contnion, smash, rodomil, tamaron, cumbush, metasestox and
methylbromide.
A study in Dallas revealed that, of 37 cases of pesticide poisoning,
almost all occurred in the home, and ingestion of liquid was the most common
exposure route (67%). These oral poisoning occurred when the child drank
improperly stored liquid agents, contacted a contaminated container or object,
or ate insecticide granules (ZWeiner and Ginsburg, 1988). Concerning
spraying OP, several studies between the years 1980-1990 in "Israel" were
reported on the toxic risks associated with aerial sprayed organophosphate in
pilot, crew, field and green house workers and kibbutz residents, including
children (Richter and Safi, 1997).
In Sri Lanka about 10,000-20,000 admissions to hospital for OPP occur
each year, of these at least 10% die. In most cases, the poisoning is
intentional (Roberts et al., 2003). In Central America, occupational poisoning
is reported to be more common than international poisoning, and deaths are
fewer (Wesseling et al., 1997).
In Washington State it was found that 44% of children of pesticide
applicator and 27% of nonfarm, rural children had detectable
organophosphorus residues (Loewenherz et al., 1997). In Costa Rica children
less than five years accounted for 39.2% pesticide poisoning cases in 1997,
and the prevalence was the same in boys and girls (leveridge, 1999).
In Nayarit State, Mexico, organophosphorous and carbamic pesticides
are used in large quantities on tobacco plantations, where up to 3000 children
and their families work. Organophosphorous and carbamic pesticides are
easily inhaled or absorbed through the skin and children may be particularly
vulnerable to pesticides because of their smaller body mass, their height and
more regular hand-mouth contact (Gamlin et al., 2007)
2.4 Pathophysiology of organophosphorus pesticide poisoning
Organophosphorus pesticides can be absorbed rapidly via all routes:
respiratory, gastrointestinal, ocular, and dermal. The effects of OP on human
physiology are multiple and complex. Organophosphorus pesticides inhibit
numerous enzymes, of which esterases seem to be the most clinically
important. The nervous system contains acetylcholinesterase which is
irreversibly phosphorylated and inhibited by OP. Inhibition of
acetylcholinesterase leads to the accumulation of acetylcholine at cholinergic
synapses, interfering with the normal function of the autonomic, somatic, and
central nervous systems (Fig. 2.1).
Fig.2.1. Pathophysiology of organophosphorus pesticide poisoning
This produces a range of clinical manifestations, known as the acute
cholinergic crisis (Eyer, 2003; Clark, 2006 and Eddleston, 2008).
Human serum contains both serum AChE and BuChE with AChE
levels are much lower (Wilson and Henderson, 1992). Therefore, serum
cholinesterase is conveniently used as a biomarker for exposures to OP
(Lopez-Carillo and Lopez-Cervantes, 1993; Hillman, 1994; Misra, 1994 and
Safi et al., 2005).
Carbamate pesticides also induce an acute cholinergic crisis through
inhibition of acetylcholinesterase. Because carbamates are structurally
different from organophosphorus compounds, acetylcholinesterase inhibited
by carbamates reversibly, allowing spontaneous reactivation and restoration
of normal nervous function. Carbamates are considered to cause milder
poisoning of shorter duration than organophosphorus pesticides. Evidence is,
however, mounting that severe toxicity and death occur with some
carbamates, in particular carbosulfan and carbofuran (Lotti, 1995).
Although very few reports mentioned poisoning cases related to
pesticides, no data are available on cholinesterase in OPP among children in
Gaza Strip. It was mentioned that pesticides are responsible for 20-30% of all
cases of poisoning in Gaza Strip including children (Nabulseya, 1993). In
addition, Richter and Safi, (1997) reported that children in Gaza Strip and
West Bank continue to have episodes of acute poisoning after accidental
pesticides ingestion. This is attributed to inadequate packaging and labeling of
pesticides and consumers' lack of knowledge about proper handling and use.
The effect of exposure to pesticides among children in a Nicaraguan
community in the path of rain water runoff from a large crop-dusting airport
was evaluated by measuring plasma cholinesterase (McConnel et al., 1999).
Mean cholinesterase activity in 17 children in the path of runoff was 2.4 IU/ml
/min, lower than the 2.9 IU/ml/min measured in a group of 43 children from an
unexposed community (difference=0.49 IU/ml/min; 95% C.I. 0.24, 0.76). Six
(35%) of the 17 exposed children had abnormally low cholinesterase levels. A
possible explanation for this physiological effect of exposure to pesticides is
the dermal absorption which may have occurred among children playing
barefoot in puddles grossly contaminated by runoff from the airport.
Gamlin et al. (2007) assessed the effect of organophosphorous and
carbamic pesticide exposure on acetylcholinesterase levels of very young
migrant Mexican tobacco workers and younger siblings. Blood samples were
collected from 160 children aged 0-14 years during harvest (exposure) and
from 62 children in their home communities 6-9 months after harvest
(baseline). Samples were tested for cholinesterase corrected for haemoglobin
and ambient temperature. Fifteen per cent of children had depression scores
ranging from -40% to 190% of their baseline levels. Thirty-three per cent of
children had depression scores of at least 15% and 86% of children were
anaemic.
Aretrospective study was conducted on Egyptian children with
Organophosphorus and carbamate poisoning (El-Naggar et al., 2009). Forty
seven patients were included. The diagnosis was confirmed by measuring
plasma cholinesterase levels. Atropine was given intravenous (0.2 mg/kg) and
repeated until secretions were controlled. Obidoxime chloride was
administered as 4-8 mg/kg/dose for children with OPP and to those in whom
the ingested material was unidentified on admission. Most of the patients
showed marked reactivation in plasma cholinesterase within several hours
and recovered completely within 24 h of admission. Complications were
observed in 17 patients (36%). Mechanical ventilatory support was required in
six patients. The duration intensive care stay was 3±2.4 days. The study
concluded that low plasma cholinesterase levels support the diagnosis of
insecticides poisoning, but no significant association is present between the
severity of poisoning and plasma cholinesterase levels.
2.5 Toxicity symptoms of organophosphorus pesticide poisoning
Organophosphorus pesticide inhibits irreversibly acetylcholinesterase
in the nervous system. This Inhibition leads to the accumulation of
acetylcholine at cholinergic synapses producing a range of clinical
manifestations, known as the acute cholinergic crisis. The particular clinical
features depends on the type of receptors and their location (Namba, 1971;
Eyer, 2003; Eddleston et al., 2006 and Karalliedde et al., 2006 ).
A. Muscarinic receptors: diarrhoea, urinary frequency, miosis, bradycardia,
bronchorrhoea and bronchoconstriction, emesis, lacrimation, salivation,
hypotension and cardiac arrhythmias.
B. Nicotinic receptors: fasciculations and muscle weakness, which may
progress to paralysis and respiratory failure, mydriasis, tachycardia and
hypertension.
C. Central nervous system: altered level of consciousness, respiratory
failure and seizures.
Zwiener and Ginsburg (1988) and Sherman (1995) reported that the
most frequent acute symptoms of OPP in children include miosis, excessive
salivation, nausea and vomiting, lethargy, muscle weakness, tachycardia,
hyporeflexia, hypertonia, and respiratory distress. Duration of symptoms
depends on the dose, with the highest doses resulting in death. Pneumonitis
developed in about one-third of poisoned children.
Wax et al., (1994) reported that pesticides exposure, even at low level,
can trigger sever reactions among persons with asthma, especially children.
However, there is almost no epidemiological data on pesticide exposure as an
independent risk factor for asthma in children. A fatality associated with
sudden irreversible bronchospasm from a pyrethrin shampoo was reported.
In his review on human health effects of pesticides, Keifer (1997)
reported that some signs and symptoms of pesticide-related health effects
include headaches, nausea, dizziness and restlessness. However, various
pesticides can also cause weakness, muscle twitching, profuse sweating, skin
irritation and even convulsions. These signs and symptoms may vary in
severity depending on the concentration, toxicity, route of exposure, and type
of pesticide.
Lifshitz et al., (1999) demonstrated that common toxicity symptoms of
OPP in young children are manifested in tearing, salivation, rhinitis, nausea,
vomiting, diarrhea, bronchoconstriction, urination, weakness, tachycardia,
headache, dizziness, confusion, tremors, cramps, convulsions, paralysis and
even death. Reigart and Roberts, (1999) pointed out that OPP can cause a range of
symptoms in adults and children, depending on the type of pesticide. For
example, commonly used organophosphorus compounds can produce
neurobehavioral effects, such as fatigue, dizziness, and blurred vision;
intestinal effects, respiratory effects such as dry throat and difficulty with
breathing; effects involving skin and mucous membranes, such as stinging
eyes, itchy skin, and a burning nose; and muscular symptom, such as
stiffness and weakness.
Knowledge, attitude, practice and toxicity symptoms associated with
pesticide use and exposure among 189 farm workers in the Gaza Strip were
assessed (Yassin et al., 2002). The most common self-reported symptoms
were burning sensation in eyes/face 119 (64.3%), dizziness 60 (32.4%),
cold/breathlessness/chest pain 52 (28.1%), itching and skin irritation 50
(27.0%) and headache 49 (26.5%).
Alarcon et al., (2005) showed that exposure to pesticides at schools
has been associated with illnesses among employees and students, although
infrequently. Rates of illness from pesticide exposure at schools have been
shown to be higher in school staff than in children because staff members are
more likely to handle pesticides. Exposures to pesticides can produce cough,
shortness of breath, nausea, vomiting, headaches, and eye irritation.
DeAnda et al., (2009) reported three recent poisoning incidents from
drifting pesticides in school children waiting at school bus stops in the San
Joaquin Valley, California. They showed that pesticides can cause both short-
term and chronic adverse health effects, including stinging eyes, breathing
difficulties, rashes, blisters, blindness, nausea, dizziness, diarrhea and even
death. Possible chronic effects include cancer, birth defects, reproductive
harm, neurological and developmental toxicity, and disruption of the endocrine
(hormonal) system. Pesticide exposure can also trigger asthma attacks. They
added that California Department of Pesticide Regulation and County
Agriculture Commissioners must take immediate steps to protect children
from the dangers of pesticides where they live and play by requiring buffer
zones around homes, schools and school bus stops and imposing steep
sanctions on applicators who put others in harm's way.
2.6 Effect of organophosphorus pesticides on body organs Although extensive research has been focused on the action of
organophosphorus pesticides on the nervous system of target and non
species Mileson et al., 1998. Little investigation has been done on their effects
on other organs particularly in children. However, some studies showed that
OP have harmful effects on body organs including liver (Yassin, 2003), kidney
(Balali-Mood and Balali-Mood (2008), blood and immune system (Forget,
1991; El-Sebae, 1993; Wesseling et al., 1997; Sungur and Guven, 2001 and
Safi et al., 2005) and respiratory organs (Eddleston et al., 2006).
2.6.1 Effect of organophosphorus pesticides on liver
Children may have an increased sensitivity to pesticides due to their
small body mass and because they differ physiologically from adults. For
instance, in newborns, liver function has not reached its full potential; thus
concentrations of enzymes needed to detoxify certain pesticides may be low
(Kifer, 1997).
Tomei et al.,(1998) reported considerable liver damage with significant
increase in AST, ALP and total bilirubin among environmental insecticides
disinfectant users in Italy. High degree of abnormal liver function may indicate
toxic effects of pesticides and the presence of pesticide residues in blood
(Amr, 1999). Pesticides residues and their metabolites in various human
tissues and fluid are indicative of past and present exposure. Altered liver
enzyme activities have been reported due to occupational exposure to OP
alone or in combination with organochlorine (Anwar, 1997).
Goel et al., (2000) also demonstrated a significant increase in the level
of various serum and liver markers such as ALP, ALT and AST due to the
effects of chloriphyrifos. Greater the degree of pesticide exposure
greater would be the levels of liver enzymes as reported in two cases
of acute endosulfan toxicity (Yavuz et al., 2007). An increase in the AST and
ALT levels was also reported in agricultural workers in "Israel" (Hernandez et
al., 2006), India (Patil et al., 2003) and selected farm workers in Pakistan
(Azmi et al., 2006).
A study reported early biochemical changes in serum enzymes after
exposure to mixture of pesticides (Hernandez et al., 2006). Altuntus et al.,
(2002) reported increased activity of ALT and AST in people engaged in
agricultural and public health programs due to the effects of Methidathion, one
of the most widely used organophosphorus compound in this program.
Bhalli et al., 2006, assessed the genotoxic effects of pesticides on
workers involved in the pesticide manufacturing industry. The exposed
subjects were 29 and the controls (unexposed) were 35 individuals from the
same area but was not involved in pesticide production. Liver enzymes,
serum cholinesterase, micronucleus assay and some haematological
parameters were used as biomarkers in the study. A statistically significant
(P<0.001) increase in levels of ALT, AST and ALP was detected in exposed
workers with respect to the control group. There was a significant (P<0.001)
decrease in the level of cholinesterase in the exposed group. Exposed
individuals exhibited cytogenetic damage with increased frequencies
(P<0.001) of binucleated cells with micronuclei and total number of
micronuclei in binucleated lymphocytes in comparison with subjects of the
control group. A decrease (P<0.001) in cytokinesis block proliferation index
similarly demonstrates a genotoxic effect due to pesticide exposure.
The frequency of plasma pesticide residues above acceptable daily
intake (ADI) and its correlation with biochemical markers for assessment of
adverse health effects in the tobacco farmers including children at district
Sawabi, Pakistan was determined (Khan et al., 2008). Total 109 males
consisting of 55 tobacco farmers exposed to pesticides and 54 controls were
included. The tobacco farmers had multiple pesticides residues above ADI in
their blood consisting of 35 (63%) methomyl; 31 (56%) thiodicarb; 34(62%)
cypermethrin; 27 (49%) Imidacloprid; 18 (32%) Methamidophos and 15 (27%)
endosulfan. Butyrylcholinesterase activity was significantly decreased in the
pesticides exposed farmers as compared to controls (P<0.001). Serum ALT,
AST, Creatine Kinase(CK), and Lactate dehydrogenase (LDH) were
significantly raised in the pesticides exposed farmers as compared to control
group (P<0.001). Total pesticides residues revealed a significant positive
correlation with AST (r=0.42), LDH (r= 0.47) and ALT (r=0.20).
2.6.2 Effect of organophosphorus pesticides on the kidneys In their studies on organophosphorus pesticides in humans, Gallo and
Lawryk, (1991), reported that OPP elevated frequently serum creatinine,
creatinine phosphokinase, and serum ALT. The fact that LDH and serum AST
do not undergo a parallel change, and that creatinine occurs almost
exclusively in the muscles, suggest that the striated muscles undergo hypoxic
damage during poisoning and exclude substantial liver involvement.
However, temporary liver damage (increased urinary urobilinogen, or delayed
excretion of bromosulphothalein) may occur.
Abend et al., (1994), reported that rhabdomyolysis is a well-known
complication of severe poisonings and appears to be also relatively frequent
in severe OP intoxication including diazinon. In the acute phase this may
cause acute renal failure and in later stages paresis if not treated correctly.
Acute renal insufficiency has been described in one patient exposed to
malathion spray (Reynolds, 1996).
Biological parameters of OP sprayers in the Gaza Strip were assessed
(Yassin, 2003). Seventy-four OP sprayers were commonly engaged in
spraying methamidophos, chlorpyrifos and dimethoate. The reference group
was represented by thirty individuals selected from the general population on
the basis of being never been exposed to pesticides. Exposure of sprayers to
different OP lowered significantly the activity of serum cholinesterase
(mean=3891±178 v 3226±152 IU/L, % difference=17.1, p=0.011).
Concentrations of serum urea and uric acid were significantly increased
(22.9±1.0 v 26.0±1.1 mg/dl, % difference=13.5, p=0.048 and 5.07±0.17 v
5.69±0.20 mg/dl, % difference=12.2, p=0.045, respectively). Serum creatinine
was increased in comparison with controls, but the change was not significant
(1.08±0.02 v 1.16±0.03 mg/dl, % difference=7.4, p=0.076. In addition, there
were disturbances in serum electrolytes including calcium and phosphorus.
Yousaf et al., (2003) reported subtle nephrotoxic changes in
occupational exposure to pesticides with higher levels of serum creatinine
and urea. Exposure to pesticide in a chemical plant also showed a
significantly higher serum creatinine concentration which supports the
subclinica kidney impairment (Kossmann et al., 2001). Attia (2006) reported
serum creatinine and urea in upper reference range in the pesticide
applicators.
A total of 85 pesticide sprayers including children in grape garden
exposed to different class of pesticides for 3 to 10 years were compared with
75 controls matched for age with respect to serum cholinesterase, serum total
protein, albumin, AST, ALT, hematological parameters and serum lipid
peroxidation (Jyotsna et al., 2003). Significant decrease was observed in
serum cholinesterase, serum total proteins, albumin and hematological
parameters viz. Hb, Hct and RBC. Significant increase in lipid peroxidation,
AST, ALT, was observed in exposed group when compared with control.
Balali-Mood and Balali-Mood, (2008) reported that acid-base and
electrolyte disturbances are common during the sever OPP. Arterial blood gas
analysis and estimation of serum electrolytes, liver and kidney function tests,
Amylase, Creatine Kinase, and Lactate dehydrogenase may be required for
the management of patients. Hypokalemia and hyperglycemia are common
and should be considered and corrected. Elevation of serum amylase and
lipase may reveal acute pancreatities. Transient elevation of liver enzyme,
hematuria, leukocyturia, and proteinuria, may be observed during nerve agent
poisoning. Blood cell count and other hematological tests may be performed
as clinically indicated. Serum urea and creatinine were found to be
significantly raised in tobacco farmers including children as compared to
controls (Khan et al., 2008).
2.6.3 Effect of organophosphorus pesticides on blood
Pesticides including OP insecticides have been shown to have
hematotoxic properties and may cause aplastic anemia, agranulocytosis,
neutropenia, and thrombopenia (Parent-Massin and Thouvenot, 1993). Far
more serious and long-term consequences have been seen in humans by
Khristeva and Mirchev, (1993). They found that both acute and chronic
exposure to toxic doses of pesticides as well as drugs and heavy metals may
induce hematologic congenital abnormalities, particularly glucose 6 phosphate
dehydrogenase deficiency and thalassemia.
Lander and Ronne, (1995), also found significant odds ratio for
leukemia among farmers. This pointed out the role of pesticides in
carcinogenesis and disruption of hematopoiesis. Genotoxicity has also been
linked to pesticides (Varona et al., 2003 and Undeger and Basaran, 2005).
Changes in hemoglobin levels as well as electrocardiograms have
been previously associated with early hexachlorocyclohexane exposure
(Srivastava et al., 1995). A similar association between RBC count and
pesticide use was also reported with hexachlorocyclohexane by Shouche and
Rathore, (1997). In addition, Casale et al., (1998) have found that pesticide
use is a significant predictor of RBC count and hematocrit and that extensive
use of pesticides significantly reduces serum complement activity.
A total of 85 pesticide sprayers exposed to different classes of
pesticides including organophosphorus insecticides for 3 to 10 years were
compared with 75 controls (Jyotsna et al., 2003). Significant decrease was
observed in serum cholinesterase and hematological parameters viz. Hb, Hct
and RBC in exposed group compared to control. The effect of OP on Hb in
exposed humans has been observed (Ray, 1992).
Hematological biomarkers in 48 farm workers exposed to OP in the
Gaza Strip were assessed (Safi et al., 2005). Results showed significant
decreases in Hb content and hematocrit value for farm workers across a shift
(14.63±1.18 g/dl and 45.58±4.64 % v 14.20±1.25 g/dl and 43.81±4.16 %,
P<0.05). However, in farm workers, White blood cell count and platelets
counts significantly elevated (6.64±1.73 x103cell/µl and 194.4±85.93
x103cell/µl v 7.44±2.01 x103cell/µl and 225.4±72.77 x103cell/µl, P<0.01 and
P=0.04, respectively) at the end of spraying day compared with the beginning
of the same spraying day.
In their cross-sectional study, Leilanie Prado-Lu, (2007) determined
associations between hematologic indices with illnesses related to pesticide
exposure among cutflower farmers in Philippines. One hundred two randomly
selected cutflower farmers including children underwent comprehensive,
personal physical health and laboratory examinations and answered a
questionnaire on work practices and illness. Results showed that illness due
to pesticides (p = 0.005) was correlated with abnormal MCV. Significant
associations were also found for hemoglobin, hematocrit, RBC, white blood
cell (WBC) and platelet count.
Rastogi et al., (2008) evaluated the health impact of spraying OP in 34
male sprayers including children in the mango belt of Malihabad, North India.
Plasma BuChE and complete blood count were assessed among sprayers
after spraying pesticides and the findings obtained were compared with those
determined in a reference group (n=18). Plasma BuChE was significantly
decreased in workers. The results indicated significant decrease in the mean
value of hemoglobin, hematocrit and platelets count; however, significantly
higher count of leukocytes was also observed in the exposed group (sprayers)
compared to that observed in the control group (P<0.05).
Chapter 3
Materials and methods 3.1 Study Design A cross-sectional design was used in the present study. 3.2 Period of the study
The study started at the beginning March 2008 to June 2009. The data
collection took around nine months. The rest of the period was necessary to
analyze, interpreting data and writing up the thesis.
3.3 Setting of the Study
The study was carried out in Gaza City, blood samples were collected
from the poisoned children admitted to El-Dorah and El-Nasser Hospitals lab
and from controls.
3.4 Study population
The target population was OP poisoned children who admitted to El-
Dorah and El-Nasser Hospitals.
3.5 Sample size
The Sample size was 104 poisoned children (58 males and 46
females) and 97 healthy children (51 males and 46 females) served as control
group aged 1-12 years.
3.6 Sampling
A total numbers of 104 blood samples were collected from poisoned
children cases, who were diagnosed according to the current WHO
diagnostic at El-Dorah and El-Nasser hospitals in Gaza City. These two
hospitals receive the poisoning cases of children in Gaza City Governorate
(54 from El-Dorah and 50 from El -Nasser). Ninty seven blood samples were
also collected from healthy children who will served as controls (Children
aged between 1-12 years).
3.7 Ethical Consideration
The necessary approval have been obtained to conduct the study from
Helsinki committee in the Gaza Strip (Annex 1 and Annex2). The approval
was issued on June, 2009. Helsinki committee is an authorized professional
body for giving permission to researchers to conduct their studies with ethical
concern in the area. One official letter of requests sent from the Islamic
University of Gaza to Palestinian Ministry of Health (MOH) to obtain approval
to conduct the study in El-Dorah and El-Nasser hospital (Annex 3). The
children sponsors were given a full explanation about the purpose of the study
and assurance about the confidentiality of the information and that the
participation was optional.
3.8 Samples collection and processing Venous blood sample (about 7 ml) was drawn by well trained medical
technologist into vacutainer tubes from the exposed and the control children.
About 2 ml blood was placed into EDTA vacutainer tube for complete blood
count. The remainder quantity of blood was left for a while without
anticoagulant to allow blood to clot. Then serum samples were obtained by
centrifugation at room temperature by Rotina 46 Hettich centrifuge, Japan at
4000 rpm/10 minutes and then samples were stored in refrigerator until
biochemical analysis .
3.9 Questionnaire interview
A meeting interview used for filling in the questionnaire which
designated for matching the study need. All interviews were conducted face to
face by the researcher. The questionnaire (Annex 4) was based on OPP
study with some modification (Yassin et al., 2002). During the study the
interviewer explained to the children sponsors any of the confused questions
that were not clear to them. Most questions were the yes/no questions, which
offer a dichotomous choice (Backestrom and Hursh-Cesar, 1981). The
questionnaire includes questions on the personal data, causes of pesticides
poisoning, domestic use of pesticides, treatment of pets (especially cats/dogs)
with pesticides, recent pesticides treatment of children with head lice and
garden or farm activities. 3.10 Biochemical analysis 3.10.1 Determination of cholinesterase activity
The procedure used for the determination of cholinesterase in serum samples
was based on the method described by Ellman et al., (1961). Principle
Cholinesterase hydrolyses butyrylthiocholine under release of butyric acid
and thiocholine. Thiocholine reduces yellow potassium hexacyanoferrate (III)
to colorless potassium hexacyanoferrate (II). The decrease of absorbance is
measured at 405 nm. Reagents
Reagent Component Concentration
Reagent 1
Pyrophosphate pH 7.6
Potassium hexacyanoferrate(III)
75 mmol/L
2 mmol/L
Reagent 2
Butyrylthiocholine
15 mmol/L
Procedure
To 1 ml of R1, 20 µL of sample and 20 µL of Dist. water were added at 37ºC
mixed, and incubated 3 minutes at room temperature then added 250 µL of
R2 mixed, read absorbance's at 405 nm, after 2 min. and start stop watch.
Read absorbance again after 1,2 and 3 min.
∆A/min = [∆A/min Sample] – [∆A/min Blank]
Calculation Calculate ∆A/min and multiply with 68500 =cholinesterase activity U/L . Normal range : 3930 – 10800 U/L . 3.10.2 Determination of serum glucose Serum Glucose was determined by glucose oxidase (GOD)/glucose
peroxidase (POD) method (Trinder,1969) using Biosystems Reagent Kits
(Spain).
Principle
Glucose in the sample originates, by means of the coupled reaction described
below, a coloured complex that can be measured by spectrophotometry.
Glucose + 2H20 + O2 GOD Gluconic acid + H202
H2O2 + Phenol +4-Aminoantipyrine (4-AP) POD Quinone + H2O Reagents
Reagent Component Concentration
A. Reagent
phosphate
Phenol
Glucose oxidase (GOD)
Peroxidase (POD)
4-AP
100 mmol/L
5 mmol/L
10 U/mL
1 U/mL
0.4 mmol/L, pH 7.5
S. Standard
Glucose Standard
100 mgl/dL
Procedure
1. Bring the working Reagents and the photometer to 370C.
2. Pipette into a cuvette:
LDH
ALT
Working Reagent
Standard (S) or sample
1.0mL
10µL
3. Mix thoroughly and incubate the cuvette for 10 minutes at room
temperature or for 5 minutes at 370C.
4. Measure the absorbance (A) of the Standard and the Sample at 500 nm
against the Blank. The colour is stable for at least 2 hours.
Calculation
A Sample
X C Standard = C Sample
A Standard
Normal range: 70 – 105 mg/dl. 3.10.3 Determination of alanine aminotransferase (ALT) activity The activity of ALT was determined according to Gella method (Gella et al.,
1985) using Biosystems Reagent Kits (Spain).
Principle
Alanine aminotransferase catalyzes the transfer of the amino group from
alanine to 2-oxoglutarate, forming pyruvate and glutamate. The catalytic
concentration is determined from the rate of decrease of NADH, measured at
340 nm, by means of the lactate dehydrogenase (LDH) coupled reaction.
Alanine + 2 – Oxoglutarate Pyruvate + Glutamate
Pyruvate + NADH +H+ Lactate + NAD+
Reagents
A Reagent: Tris 150mmol/L, L-alanine 750mmol/L, lactate dehydrogenase
>1350 U/L, pH 7.3.
∆A/min x = U/L ε x I x VS
B Reagent: NADH 1.3 mmol/L, 2-oxoglutarate 75 mmol/L, Sodium hydroxide
148 mmol/L, sodium azide 9.5 g/L.
C Reagent (code11666): Pyroxial phosphate 10 mmol/l. 5mL. Reagent preparation
Working Reagent: Pour the contents of the Reagent B into the Reagent A
bottle. Mix gently. Other volumes can be prepared in the proportion: 4 mL
Reagent A + 1 mL Reagent B. Stable for 2 months at 2-8 0C.
Working Reagent with Pyridoxal Phosphate: Mix as follows: 10 mL of Working
Reagent + 0.1 mL of Reagent C (cod 11666). Stable for 6 days at 2-8 0C.
Procedure
1. Bring the Working Reagent and the instrument to reaction temperature.
2. Pipette into a cuvette:
Reagent temperature 37 0C 30 0C
Working reagent 1 mL 1 mL
Sample 50 µL 100 µL
3. Mix and insert the cuvette into the photometer. Start the stopwatch.
4. after 1 minute, record initial absorbance and at 1 minute intervals thereafter
for 3 minutes.
5. Calculate the difference between consecutive absorbances, and the
average absorbance difference per minute (∆A/min).
Calculation Alanine aminotransferase concentration in the sample is calculated using the
following general formula:
The molar absorbance (ε) of NADH at 340 nm is 6300, the light path (l) is 1
cm, the total reaction volume (Vt) is 1.05 at 370C and 1.1 at 300C, the sample
volume (Vs) is 0.05 at 370C and 0.1 at 300C, and 1 U/L are 0.0166 µkat/L.
Vt x 106
MALATE
The following formulas are deduced for the calculation of the catalytic
concentration:
37 C 30 C
∆A/min
x 3333 = U/L x 1746 = U/L
x 55.55 = µkat/L x 29.1 = µkat/L
Normal range for ALT up to 40 U/L.
3.10.4 Determination of aspartate aminotransferase (AST) activity
The activity of AST was determined according to Gella method (Gella et al.,
1985) using Biosystems Reagent Kits (Spain).
Principle
Aspartate aminotransferase catalyzes the transfer of the amino group from
aspartate to 2-oxoglutarate, forming oxalacetate and glutamate. The catalytic
concentration is determined from the rate of decrease of NADH, measured at
340 nm, by means of the malate dehydrogenase (MDH) coupled reaction.
Aspartate + 2Oxoglutarate Oxalacetate + Glutamate
Oxalacetate + NADH + H+ Malate + NAD+
Reagents
A Reagent: Tris 121mmol/L, L-aspartate 362mmol/L, malate
dehydrogenase>460U/L, lactate dehydrogenase >660U/L, Sodium hydroxide
255mmol/L, pH 7.8.
B Reagent: NADH 1.3mmol/L, 2-oxoglutarate 75mmol/L, Sodium hydroxide
148mmol/L, sodium azide 9.5g/L.
C Reagent (code11666): Pyroxial phosphate 10mmol/l. 5mL.
AST
∆A/min x = U/L ε x I x VS
Reagent preparation
Working Reagent: Pour the contents of the Reagent B into the Reagent A
bottle. Mix gently. Other volumes can be prepared in the proportion: 4mL
Reagent A+1mL Reagent B. Stable for 2 months at 2-80C.
Working Reagent with Pyridoxal Phosphate: Mix as follows: 10mL of Working
Reagent + 0.1mL of Reagent C (cod 11666). Stable for 6 days at 2-80C. Procedure
1. Bring the working reagent and the instrument to reaction temperature.
2. Pipette into a cuvette:
Reagent temperature 370C 300C
Working reagent 1mL 1mL
Sample 50µL 100µL
3. Mix and insert the cuvette into the photometer. Start the stopwatch.
4. After 1 minute, record initial absorbance and at 1 minute intervals thereafter
for 3 minutes.
5. Calculate the difference between consecutive absorbance, and the average
absorbance difference per minute (∆A/min).
Calculation
Aspartate aminotransferase concentration in the sample is calculated using
the following general formula:
The molar absorbance (ε) of NADH at 340 nm is 6300, the light path (l) is 1
cm, the total reaction volume (Vt) is 1.05 at 370C and 1.1 at 300C, the sample
volume (Vs) is 0.05 at 370C and 0.1 at 300C, and 1 U/L are 0.0166µkat/L. The
following formulas are deduced for the calculation of the catalytic
concentration.
Vt x 106
ALP
370C 300C
∆A/min
x 3333 = U/L x 1746 = U/L
x 55.55 = µkat/L x 29.1 = µkat/L
Normal range, for AST up to 38 U/L.
3.10.5 Determination of alkaline phosphatase (ALP) activity The activity of ALP was measured according to Rosalki method (Rosalki et
al., 1993). using Biosystems Reagent Kits (Spain).
Principle
Alkaline phosphatase catalyzes in alkaline medium the transfer of the
phosphate group from 4-nitrophenylphosphate to 2-amino-2-methyl-1-
propanol (AMP), liberating 4-nitrophenol. The catalytic concentration is
determined from the rate of 4-nitrophenol formation, measured at 405 nm.
4 – Nitrophenylphosphate + AMP AMP – phosphate + 4 – Nitrophenol
Reagents
A Reagent: 2-Amino-2-methyl-1-propanol 0.4 mol/L, zinc sulfate 1.2 mmol/L,
N-hydroxyethyl ethylene diaminetriacetic acid 2.5 mmol/L, magnesium acetate
2.5 mmol/L, pH 10.4.
B Reagent: 4-Nitrophenylphosphate 60 mmol/L.
Reagent preparation
Working Reagent
- Cod. 11592 and 11593: Transfer the contents of one Reagent B vial into a
Reagent A bottle. Mix gently. Other volumes can be prepared in the
proportion: 4 mL Reagent A + 1 mL Reagent B. Stable for 2 months at 2-80C. - Cod. 11598: Transfer 25 mL of one Reagent B vial into a Reagent A bottle.
Mix gently. Other volumes can be prepared in the proportion: 4mL Reagent A
+ 1 mL Reagent B. Stable for 2 months at 2-80C.
Procedure
1. Bring the working reagent and the instrument to reaction temperature.
2. Pipette into a cuvette:
Working reagent 1mL
Sample 20µL
3. Mix and insert the cuvette into the photometer.
4. Record initial absorbance and at 1 minute intervals thereafter for 3 minutes.
5. Calculate the difference between consecutive absorbencies, and the
average absorbance difference per minute (∆A/min).
Calculation
Alkaline phosphatase catalytic concentration in the sample is calculated
using the following general formula:
The molar absorbance (ε) of 4-nitrophenol at 405 nm is 18450, the light path
(l) is 1 cm, the total reaction volume (Vt) is 1.02, the sample volume (Vs) is
0.02, and 1 U/L are 0.0166 µkat/L. The following formulas are deduced for the
calculation of the catalytic concentration:
∆A/min
x 2764 = U/L
x 46.08 = µkat/L
Normal range up to 645 U/L.
3.10.6 Determination of urea Urea determination is based upon the cleavage of urea by urease according
to Burtis assay (Burtis et al., 1994) using Biosystems Reagent Kits (Spain).
∆A/min x = U/L Vt x 106
ε x I x VS
nitroprusside
urease
Principle
Urea in the sample consumes, by means of the coupled reaction described
below, NADH that can be measured by spectrophotometry.
Urea + H2O 2NH4+ + CO2
NH4+ + Salicylate + NaCIO Indophenol
Reagents A Reagent: Tris 100mmol/L, 2-oxoglutarate 5.6mmol/L, urease >140U/mL,
glutamate dehydrogenase >140U/mL, ethyleneglycol 220g/L, sodium azide9.5
g/L, pH8.0.
B Reagent: NADH 1.5 mmol/L, sodium azide 9.5g/L.
S Glucose/Urea/Creatinine Standard. Glucose 100mg/dL, urea 50mg/dL
(8.3mmol/L, BUN 23.3mg/dL), creatinine 2mg/dL. Aqueous primary standard.
Reagent preparation
Working Reagent: transfer the content of one reagent B vial into a reagent A
bottle. Mix gently. Other volumes can be prepared in the proportion: 4mL
Reagent A+1mL Reagent B. stable for 2 months at 2-80C.
Procedure
1. Bring the working Reagents and the photometer to 370C.
2. Pipette into a cuvette:
Working Reagent
Standard (S) or sample
1.5mL
10µL
3. Mix and insert the cuvette into the photometer. Start stopwatch.
4. Record the absorbance at 340 nm after 30 seconds (A1) and after 90
seconds (A2).
Calculation
The urea concentration in the sample is calculated using the following general
formula:
(A1-A2) Sample
X C Standard X Sample dilution factor = C Sample
(A1-A2) Standard
If the Urea Standard provided has been used to calibrate:
Serum and plasma
(A1-A2) Sample
(A1-A2) Standard
x 50 = mg/dL urea
x 23.3 = mg/dL BUN
x 8.3 = mmol/L urea
Normal range:10 –45 mg/dl.
3.10.7 Determination of creatinine Serum creatinine was determined kinetically using Biosystems Reagent Kits
(Spain) and following their instruction manual described by Fabiny and
Ertingshausen, (1971).
Principle Creatinine in the sample reacts with picrate in alkaline medium forming a
colored complex. The complex formation rate is measured in a short period to
avoid interferences.
Reagents
A Reagent Picric acid 25mmol/L.
B Reagent Sodium hydroxide 0.4mol/L, detergent.
S Glucose / urea / creatinine standard. Glucose 100mg/dl, urea 50mg/dl,
creatinine 2mg/dl (177 µmol/L). Aqueous primary standard.
Reagents preparation
Standard (S) is provided ready to use.
Working reagent: mix equal volumes of reagent A and reagent B. mix
thoroughly. Stable for 1 month at 2- 80C.
Procedure
1. Bring the Working Reagent and the photometer to 370C.
2. Pipette into a cuvette:
Working Reagent
Standard (S) or Sample
1.0mL
0.1mL
3. Mix and insert cuvette into the photometer. Start stopwatch.
4. Record the absorbance at 500 nm after 30 seconds (A1) and after 90
seconds (A2).
Calculation
The creatinine concentration in the sample is calculated using the following
general formula:
(A2 – A1) Sample
x C Standard x Sample dilution factor = C Sample
(A2 – A1) Standard
If the creatinine standard provided has been used to calibrate:
Serum and plasma
(A2 – A1) Sample
(A2 – A1) Standard
x 2 = mg/dL creatinine
x 177 = µmol/L creatinine
Normal range from: 0.4 –1.0 mg/dl.
3.10.8 Determination of Total protein Determination of serum proteins by means of the Gornall, (1949) reaction
using BioSystems kit, Spain. . Principle
Protein in the sample reacts with copper (II) ion in alkaline medium forming a
coloured complex that can be measured by spectrophotometry.
Reagents
A. Reagent 1 x 50 mL 2 x 250 mL 1 x 250 mL 1 x 1 L
S. Standard 1 x 5 mL 1 x 5 mL 1 x 5 mL 1 x 5 mL Reagent composition
A. Reagent. Copper (II) acetate 6 mmol/L, potassium iodide 12 mmol/L,
sodium hydroxide 1.15 mol/L, detergent.
S. Protein Standard. Bovine albumin. Concentration is given on the label.
Concentration value is traceable to the Standard Reference Material 927
(National Institute of Standards and Technology, USA).
Procedure
1. Bring the Working Reagent and the photometer to 370C.
2. Pipette into a cuvette:
Working Reagent
Standard (S) or Sample
1.0mL
0.2mL
2. Mix thoroughly and let stand the cuvette for 1 minute at room temperature.
3. Read the absorbance (A) of the Standard and the Sample at 545 nm
against the Blank. The colour is stable for at least 2 hours.
Calculations
The protein concentration in the sample is calculated using the following
general formula:
A Sample
X C Standard = C Sample
A Standard
Normal range: 6.4-8.3 g/dL
Concentrations are lower in child. Plasma total protein concentration is 2 to 4
g/L higher due to the presence of fibrinogen as well as some other trace
proteins2.
3.10.9 Determination of albumin Determination of serum albumin with bromocresol green using BioSystems
kit, Spain (Doumas, 1971).
Principle
Albumin in the sample reacts with bromocresol green in acid medium forming
a coloured complex that can be measured by spectrophotometry1.
Contents
A. Reagent 2 x 250 mL 1 x 250 mL
S. Standard 1 x 5 mL 1 x 5 mL
Reagent
A. Reagent: Acetate buffer 100 mmol/L, bromocresol green 0.27 mmol/L,
detergent, pH 4.1.
S. Albumin Standard: Bovine albumin. Concentration is given on the label.
Concentration value is traceable to the Standard Reference Material 927
(National Institute of Standards and Technology, USA).
Procedure
1. Bring the Working Reagent and the photometer to 370C.
2. Pipette into a cuvette:
Working Reagent
Standard (S) or Sample
1.0mL
0.1mL
2. Mix thoroughly and let stand the cuvette for 1 minute at room temperature.
3. Read the absorbance (A) of the Standard and the Sample at 630 nm
against the Blank. The colour is stable for 30 minutes.
Calculations
The albumin concentration in the sample is calculated using the following
general formula:
A Sample
X C Standard = C Sample
A Standard
Normal range: 3.8-5.4 g/dL 3.10.11 Determination of Globulin Globulin was calculated according the following formula:
Globulin= Total protein - Albumin
3.10.12 Determination of serum electrolytes A full automated Nova 10 electrolyte Analyzer instrument was used to
determine sodium, potassium, calcium electrolyte.
3.10.13 Determination of serum phosphorus Serum phosphorus was determined by phosphomolybdate U.V. method
(Farrell, 1984) using Labkit kit, Spain.
Principle
Direct method for determining inorganic phosphate. Inorganic phosphate
reacts in acid medium with ammonium molybdate to form a
phosphomolybdate complex with yellow color. The intensity of the color
formed is proportional to the Pi concentration in the sample.
Reagents
Reagent Component Concentration
Reagent 1
(Molybdic)
Phosphorus Cal
Ammonium molybdate
Sulphuric acid (SO4H2)
Detergents
Phosphorus aqueous
primary calibrator
0.40 mM
210 mM
5 mgldlL
Reagent preparation
Reagent and standard are provided ready to use. Procedure
1. Bring the reagent to room temperature.
2. Pipette into cuvette:
Blank Standard Sample
Reagent .1
calibrator
Sample
1.0mL
___
___
1.0mL
10µL
___
1.0mL
___
10µL
3. Mix thoroughly and incubate for10 minutes at room temperature (16-250C)
or for 5 minutes at 370C.
4. Measure the absorbance (A) of the Standard and the Sample at 340 nm
against the Blank. The color is stable for at least 30 minutes. Calculation
Serum phosphorus concentration in the sample is calculated using the
following general formula:
(A sample / A standard) X 5 C Standard = C Sample
Normal range: 4.0 – 7.0 mg/dl.
3.11 Hematological analysis A complete system of reagents of control and calibrator, Cell-Dyn 1700 was
used to determined complete blood count (CBC) of children in El-Dorah and
El-Nasser laboratory Hospitals (ABBOTT laboratories, 2001).
3. 12. Statistical analysis Data were analyzed using Statistical Package of Social Sciences (SPSS)
system (version 13.0). The following statistical tests were applied:
• Frequency distributions
• Independent-samples t-test
• Pearson's correlation test
The percentage difference was calculated according to the formula:
Percentage difference= mean of cases – mean of controls / mean of controls
x 100.
Probability values (p) were obtained from the student’s table of ‘t’ and
significance was at p < 0.05. Range as minimum and maximum values was
used.
Microsoft Excel version 6 was used for graph plotting.
Chapter 4
Results 4.1 General characteristics of the study population
The present study is a cross sectional included 201 children: 97
controls; 51 males and 46 females and 104 OP poison cases; 58 males and
46 females (Figure 4.1).
5146
5846
05
10152025303540455055
Frequency
control case
Figure 4.1. Distribution of the study population by gender
MaleFemale
The cases were chosen from El-Dorah and El-Nasser Hospitals which are the
two hospitals that receive the poisoning cases of children in Gaza city. As
shown in figure 4.2, 54 cases were from El-Dorah hospital (28 males and 26
females) and 50 cases were from El–Nasser hospital (30 males and 20
females).
30; 29%
20; 19%28; 27%
26; 25%
Male Female
The average age of the controls was 5.8±2.6 years whereas that of cases was
4.3±2.5 years (Figure 4.3).
5.8
4.3
0123456789
1011
Mean age (year)
Control Case
Figure 4.3. Mean age of the controls and the cases
Figure 4.2. Distribution of the cases (n=104) by El-Dorah and El-Nasser Hospitals
El Nasser El Dorah
4.2 Questionnaire interview A meeting interview with sponsors of poisoned children (n=104) was
used for filling in the questionnaire. The total response for the questionnaire
interview was 46.2% (n = 48). Table 4.1 illustrates the possible causes of
pesticides poisoning among children as reported by their sponsors. Eating of
poisoned biscuits, bread or meat were commonly contributed to the poisoning
cases among children 21 (43.8%) followed by exposing to field and garden
sprayed pesticides 7 (14.6%) and then by drinking pesticides in stored bottles
5 (10.4%). However, unknown cause of pesticide poisoning among children
was reported to be 6 (12.5%).
Table 4.1. Causes of pesticides poisoning among children as reported by their
sponsors (n=48).
Cause of pesticides poisoning NO. % Eating of: Freshly sprayed fruit Freshly sprayed vegetables Poisoned biscuits, bread or meat Drinking of: Tap water from empty pesticides bottles Pesticide stored in bottles Deliberate pesticide poisoned milk Field and garden sprayed pesticides Unknown
2 2
21
3 5 2 7 6
4.2 4.2
43.8
6.3 10.4 4.2
14.6 12.5
Domestic use of pesticides, treatment of pets (especially cats/dogs) with
pesticides and recent treatment of children with head lice as reported by
children sponsors (n=48) is presented in Table 4.3. Large number of children
sponsors reported the use pesticides in their homes 31 (64.6%) to combat
insects. Only 2 (4.2%) claimed the use of pesticides for treatment of pets
(especially cats/dogs). A total of 4 (8.3%) admitted the recent treatment of
their children with head lice.
Table 4.2. Domestic use of pesticides, treatment of pets (especially cats/dogs) with
pesticides, recent pesticides treatment of children with head lice as reported by
children sponsors (n=48)
Variable NO. % Domestic use of pesticides
Yes No
Treatment of pets (especially cats/dogs) with pesticides
Yes No
Recent treatment of children with head lice Yes No
31 17
2 46
4
44
64.6 35.4
4.2 95.8
8.3
91.7
Table 4.3 shows that 18 (37.5%) of children sponsors have garden or farm;
the majority of them 12 (66.7%) said that they spraying their gardens or farms
in the presence of their children and 13 (72.2%) of them reported that they did
not store pesticide bottles in safe place i.e. easy access for children to reach.
Such garden or farm activities will no doubt put children at high risk of
pesticide poisoning. Table 4.3. Garden or farm activities as reported by children sponsors (n=48). Variable NO. % Having garden or farm
Yes No Spraying garden or farm in the presence of your children Yes No Storage pesticide bottles in safe place Yes No
18 30
12 6
5 13
37.5 62.5
66.7 33.3
27.8 72.2
4.3 Diagnosed symptoms Diagnosed symptoms associated with OPP among children (n=104)
are listed in Table 4.4. The most common diagnosed symptoms were pin
point pupils 53 (51.0%), vomiting 46 (44.2%), drawsy 42 (40.4%), conscious
40 (38.5%) and convulsions 30 (28.8%), on the other hand, the least
diagnosed symptoms were salivation 9 (8.7%) followed by asthma and
respiratory secretion 12 (11.5%) each. Table 4.4. Diagnosed symptoms among poisoned children (n=104) Diagnosed symptoms No. %
Abdominal pain Convulsions Conscious Vomiting Dizziness Drawsy Headache Pin point pupils Cough Asthma Salivation Respiratory secretion Reported two or more symptoms
22 30 40 46 22 42 16 53 16 12 9
12 96
21.2 28.8 38.5 44.2 21.2 40.4 15.4 51.0 15.4 11.5 8.7
11.5 92.3
4.4 Biochemical analysis 4.4.1 Serum cholinesterase
The average of serum cholinesterase levels in the controls and the
cases is presented in Table 4.5. The mean enzyme activity in cases was
significantly lower than that in controls showing percentage difference of
69.9% (2004.3 ± 1181.9 v 6659.8 ± 1199.2 u/l, P=0.000).
Table 4.5. Serum Cholinesterase enzyme activity of the cases and the controls
Enzyme
Control (n=97)
mean±SD
Case (n=104)
mean±SD
%
difference
t-value
P-
value Cholinesterase
(U/L) Range (min – max)
6659.8±1199.2
(4322 - 9273)
2004.3±1181.9
(251 - 3803)
-69.9
-27.71
0.000
All values were expressed as mean ± SD, P<0.05: significant
Figure 4.4 illustrates cholinesterase activity among the most common
diagnosed symptoms in organophosphorus poisoned children and in the
healthy controls in Gaza City. The lowest enzyme activity was registered in
drawsy (1269±772 u/l) whereas the highest activity was recorded for
convulsion (1946±1346 u/l).
6659.8
19461411 1638
1269 1490
0
1000
2000
3000
4000
5000
6000
7000
Cholinestrase activity (U
/L)
Control Convulsion Conscious Vomitting Drawzy Pin pointpupils
Diagnozed symptoms
Figure 4. 4 cholinestrase activity among the most common diagnosed symptoms in the OP poisoned children and in the healthy contols in Gaza City.
Table 4.6, shows the mean differences of cholinesterase activities among
patient with and without the most common diagnosed symptoms. Changes in
the mean activity of the enzyme was significantly associated with drawsy,
conciuos and pin piot (p=0.001, 0.024 and 0.030, respectively). However, no
significance change was found in cholinesterase activities in convulsion and
vomiting (p=0.195 and 0.493, respectively).
Table 4.6. Mean differences of cholinesterase activities among patients with and
without the most common diagnosed symptoms(n=104).
Diagnosed symptoms Cholinesterase(U/L)
Mean±SD
Range (U/L)
t P value
Convulsion Yes (n=30) No (n=74)
1946±1346 1632±1002
251-3803
658 - 3696
-1.305
0.195
Conscious Yes (n=40) No (n=64)
1411±811
1917±1235
685-3300 251-3803
2.299
0.024 Vomiting
Yes (n=46) No (n=58)
1638±1282 1790±968
251-3803 650-3560
0.689
0.493
Drawsy Yes (n=42) No (n=62)
1269±772
2029±1208
650-2928 251-3803
3.604
0.001
Pin point Yes (n=53) No (n=51)
1490±770
1964±1351
251-2966 251-3803
2.206
0.030
All values were expressed as mean ± SD, P>0.05: non significant, P<0.05: significant 4.4.2 Serum glucose
Table 4.7 pointed out the means of serum glucose levels in the controls
and the cases. There was a statistically significant increase in glucose level of
the cases compared to the controls (135.9±44.0 v 69.5±13.1 mg/dl, %
difference=95.5, P=0.000).
Table 4.7: Serum glucose of the controls and the cases
Tests
Control (n=97)
mean±SD
Case (n=104)
mean±SD
% difference
t-value
P-value
Glucose level
(mg/dl) Range (min – max)
69.5±13.1
(65 -119)
135.9±44.0
(57 - 280)
95.5
8.461
0.000
All values were expressed as mean ± SD, P<0.05: significant
4.4.3 Liver enzyme activities The mean activities of some liver enzymes in the controls and the
cases are shown in table 4.8. The average levels of serum ALT and AST and
ALP in the cases (16,6±6.9, 19.8±8.5 u/l and 319.0±114.3 u/l, respectively)
were significantly higher than those in the controls (11.2±2.6, 13.6±4.2 u/l and
245.2±70.1 u/l, respectively) with percentage differences of 48.2, 45.6 and
30.1, respectively and p=0.000. Table 4.8. Serum liver enzyme of the case study and the controls
Liver enzyme (U/L)
Control (n=97)
mean±SD
Case (n=104)
mean±SD
%
difference
t-
value
P-
value
ALT Range (min – max)
11.2±2.6 (8 -18)
16.6±6.9 (7 - 33)
48.2 7.240 0.000
AST Range (min – max)
13.6±4.2 (4 - 24)
19.8±8.5 (10 - 52)
45.6 6.548 0.000
ALP Range (min – max)
245.2±70.1 (117 - 405)
319.0±114.3(120 - 677)
-30.1 5.470 0.000
ALT: Alanine Aminotransferase, AST: Aspartate Aminotransferase, ALP: Alkaline
Phosphatase. All values were expressed as mean ± SD, P<0.05: significant
4.4.4 protein profile 4.4.4.1 Non- protein nitrogen constituents
The mean levels of urea and creatinine (indicators of kidney function)
of the controls and the cases are presented in Table 4.9. Urea and creatinine
levels were increased in the cases compared to the controls (21.0±9.3 v
15.2±5.0 mg/dl, % difference=38.2 and 0.50±0.13 v 0.39±0.11 mg/dl, %
difference=28.2, repectively). Such increase was statistically significant
(P=0.000). Table 4.9. Serum kidney function of the case study and the controls
Renal parameter
(mg/dl)
Control (n=97)
mean±SD
Case (n=104)
mean±SD
% difference
t-value
P-
value
Urea Range (min – max)
15.2±5.0 (7 - 31)
21.0±9.3 (9 - 67)
38.2 5.492 0.000
Creatinine Range (min – max)
0.39±0.11 (0.2 - 0.7)
0.50±0.13 (0.3 - 1.0)
28.2 6.346 0.000
All values were expressed as mean ± SD, P<0.05: significant
4.4.4.2 Protein nitrogen constituents The mean levels of total protein, albumin and globulin in serum of the
cases and the controls are given in Table 4.10. There were significant
decreases in the averages of total protein, albumin and globulin
concentrations in the cases compared to the controls showing % differences
of 8.3, 2.8 g/dl and 21.8, respectively (5.5±0.80, 3.5±0.39 and 1.97±0.68 g/dl
v 6.0±0.74, 3.6±0.57 and 2.52±0.72 g/dl with p=0.000, 0.046 and 0.000,
respectively).
Table 4.10. Serum protein profile (Total protein, albumin and globulin) of the cases
and the controls.
Protein profile (g/dl)
Control (n=97)
mean±SD
Case study (n=104)
mean±SD
% difference
t-value
P-
value
Total Protein Range (min – max)
6.0±0.74 (4.0 - 7.2)
5.5±0.80 (4.0 - 6.6)
-8.3 -5.633 0.000
Albumin Range (min – max)
3.6±0.57 (2.0 - 4.8)
3.5±0.39 (2.8 - 7.4)
-2.8 -2.010 0.046
Globulin Range (min – max)
2.52±0.72 (0.1 - 4.9)
1.97±0.68 (0.8 - 3.1)
-21.8 -5.542 0.000
All values were expressed as mean ± SD, P<0.05: significant 4.4.5 Serum electrolytes
Table 4.11, shows the averages serum electrolytes of the cases and
the controls. Potassium and phosphorus concentrations were decreased
significantly in cases compared to controls (4.0±0.45 v 4.3±0.46meq/l, %
difference=7.0 and 4.1±0.79 v 4.7±1.2 mg/dl, % difference=12.8 p=0.000,
respectively). On the other hand, sodium and calcium showed not significant
increase (139.7±3.5 v and 139.4±4.1 meq/l, % difference=0.2, p=0.596 and
9.6±0.89 v 9.4±0.69 mg/dl, % difference=2.1, p=0.068, respectively).
Table 4.11. Serum electrolytes of the case study and the controls
electrolyte
Control (n=97)
mean±SD
Case study (n=104)
mean±SD
% difference
t-value
P-
value
Sodium (meq/l) Range (min – max)
139.4±4.1 (130-150)
139.7±3.5 (131-147)
0.2 0.531 0.596
Potassium (meq/l) Range (min – max)
4.3±0.46 (3.5-5.1)
4.0±0.45 (2.6-5.0)
-7.0 -5.114 0.000
Calcium (mg/dl) Range (min – max)
9.4±0.69 (8.2-11.0)
9.6±0.89 (6.8-12.1)
2.1 1.833 0.068
Phosphorus (mg/dl) Range (min – max)
4.7±1.2 (2.3-7.0)
4.1±0.79 (2.6-6.5)
-12.8 -3.812 0.000
All values were expressed as mean ± SD, P>0.05: non significant, P<0.05: significant
4.4.6 Hematological parameters
Table 4.12 demonstrates total white blood cells count, differential and
blood platelets in the cases and the controls. White blood cell count was
markedly elevated in cases compared to the controls (14.8±7.0 v 7.5±1.5
X103cell/µl, % difference=97.3, p=0.000). For differential white blood cells,
lymphocytes were also significantly increased in the cases than the controls
(52.3±17.1 v 39.4±6.8 %, % difference=32.7, p=0.000) whereas neutrophils
were singnificantly lower in the cases than the controls (40.4±17.4 v 49.1±7.4
%, % difference=17.8, p=0.000). Like white blood cells, blood platelets were
significantly increased in the cases compared to the controls (398.3±163.6 v
307.2±59.9 X103cell/µl, % difference=29.7, p=0.000).
Table 4.12. White blood cells and blood platelets of the cases and the controls
CBC profiles
Control (n=97)
mean±SD
Case study (n=104)
mean±SD
% difference
t-value
P-
value
WBCs (X103cell/µl) Range (min – max)
7.5±1.5 (4.9 - 11.7)
14.8±7.0 (4.8 - 39.6)
97.3 9.916 0.000
Lymphocyte % Range (min – max)
39.4±6.8 (20.0 – 52.0)
52.3±17.1 (19.7 - 82.1)
32.7 6.905 0.000
Neutrophile % Range (min – max)
49.1±7.4 (30.0 - 70.0)
40.4±17.4 (10.2 - 70.0)
-17.8 -4.590 0.000
PLT (X103cell/µl) Range (min – max)
307.2±59.9 (137 - 456)
398.3±163.6(54 – 978)
29.7 5.173 0.000
WBCs: white blood cells, PLT: Blood platelets. All values were expressed as mean ± SD,
P<0.05: significant
Primary and secondary blood indices of the cases and the controls are
demonstrated in Table 4.13. For the primary blood indices, the mean of red
blood cell count was not significantly changed in cases compared to controls
(4.6±0.7 v 4.4±0.5 X106cell/µl, % difference=4.5, p=0.060). However,
hemoglobin and hematocrit were significantly decreased in the cases
compared to the controls recording % differences of 10.0 each (10.8±1.1 and
33.4±3.7 v 12.0±0.87 and 37.1±2.4 %, p=0.000). Secondary blood indices
including MCV, MCH and MCHC were also found to be lower in cases
compared to controls registering % differences of 13.0, 13.4 and 3.0,
(73.16±8.33 fl, 23.61±2.76 pg, 32.28±2.10 g/dl V 84.13±4.32 fl,
27.25±1.53pg, 32.39±0.84g/dl, p=0.000, 0.000, and 0.637, respectively).
Table 4.13. Primary secondary blood indices of the cases and the controls CBC profiles
Control (n=97)
mean±SD
Case study (n=104)
mean±SD
% difference
t-value
P-
value
RBCs count
(X106cell/µl) Range (min – max)
4.4±0.5 (3.4 - 5.4)
4.6±0.7 (3.6 - 5.7)
4.5 1.243 0.060
Hb g/dl Range (min – max)
12.0±0.87
(10.5-15.0)
10.8±1.1
(7.8 -12.7)
-10.0 9.032 0.000
Hct % Range (min – max)
37.1±2.4 (31.0 - 45.8)
33.4±3.7 (26.1 - 40.4)
-10.0 8.276 0.000
MCV fl Range (min – max)
84.13±4.32 (74.3 - 100)
73.16±8.33 (53.6 - 96.1)
-13.0 11.59 0.000
MCH pg Range (min – max)
27.25±1.53 (23.7 - 32.4)
23.61±2.76 (18.1 - 28.6)
-13.4 11.46 0.000
MCHC g/dl Range (min – max)
32.39±0.84 (30.2 - 33.9)
32.28±2.10 (27.4 - 39.6)
-0.3 0.472 0.637
RBCs: Red blood cells, Hb: Hemoglobin, Hct: Heamtocrit, MCV: Mean corpuscular volume,
MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration.
All values were expressed as mean ± SD, P>0.05: non significant, P<0.05: significant
4.4.7 Association of cholinesterase with some parameters
As indicated in Table 4.14, Pearson's correlation test showed negative
correlation between cholinesterase and glucose levels (r=-0.321). This
correlation reached statistically significant level (p=0.001). On the other hand,
positive correlation between cholinesterase and phosphorus or total protein
was also achieved (r=0.234 and 0.133, respectively). This correlation was
significant for phosphorus (p=0.017) and not significant for total protein
(p=0.179). Table 4.14. Pearson's correlation between serum cholinesterase and serum glucose,
phosphorus and total protein
Enzyme Parameter Mean±SD Pearsons correlation
P value
Cholinesterase
(mean±SD) 2004.3±1181.9
Glucose
135.9±44.0 -0.321 0.001
Phosphorus 4.1±0.79 0.234 0.017
Total protein 5.5±0.80 0.133 0.179
P>0.05: non significant, P<0.05: significant As shown in Table 4.15, Pearson's correlation test revealed negative
correlation between cholinesterase and ALT, AST or ALP activities (r=-0.291,
-0.210 and 0.014, respectively). This correlation reached statistically
significant level for both ALT and AST (p=0.003 and 0.032, respectively), but
not for ALP (p=0.890).
Table 4.15. Pearson's correlation between serum cholinesterase and serum AST,
ALT and ALP
Enzyme Liver enzyme (U/L)
Mean±SD Pearsons correlation
P value
Cholinesterase(U/L)
(mean±SD) 2004.3±1181.9
ALT
16.6±6.9
0.291
0.003
AST
19.8±8.5
0.210 0.032
ALP
319.0±114.3
0.014 0.890
ALT: Alanin aminotransaminase, AST: Aspartat aminotransaminase, ALP: Alkaline phosphatase. P>0.05: non significant, P<0.05: significant
As illustrated in Table 4.16, Pearson's correlation test revealed negative
correlation between cholinesterase and urea or creatinine levels (r=-0.079 and
0.212, respectively). This correlation was significant for creatinine (p=0.030),
but not for urea (p=0.426).
Table 4.16. Pearson's correlation between serum cholinesterase and serum Urea
and Creatinine
Enzyme Parameter Mean±SD Pearsons correlation
P value
Cholinesterase
(mean±SD) 2004.3±1181.9
Urea
21.0±9.3
0.079 0.426
Creatinine 0.5±0.13
0.212 0.030
P>0.05: non significant, P<0.05: significant
Chapter 5 Discussion
Data on OPP in the Gaza Strip are limited to few published studies on
farm workers used such highly toxic compounds. However, till now no
published data are available on OPP in children in the Gaza Strip. Therefore,
the present is the first to assess OPP and the associated risk factors among
children in Gaza city, Gaza Strip. This will be useful in prevention and
treatment strategies applied to children who may have higher exposure to
pesticides than other residents living in the same contaminated environment.
The main environmental problems of concern in the Gaza Strip include
excessive use of pesticides in the agricultural sector (Safi, 1998 and Yassin et
al., 2002), inadequate disposal and management of solid wastes (Palestinian
Environmental Authority, 1995), and improper design of sewage network
(Tubail et al., 2004 and Yassin et al., 2008).
The present results revealed a relatively low response rate for the
questionnaire interview with sponsors of the poisoned children. This could be
attributed to the fear of the child's sponsor from police interrogation as well as
to the fear of child's mother from her husband to blame her and to accuse her
to be the responsible for this act, and this may lead to their divorce.
As reported by children sponsors, eating of poisoned biscuits, bread or
meat were commonly contributed to the poisoning cases among children in
the Gaza Strip followed by exposing to field and garden sprayed pesticides
and then by drinking pesticides in stored bottles. Surprisingly, two mothers
admitted two cases of deliberate pesticide poisoned in milk. It was astonishing
when the mothers claimed that children fathers introduced pesticides in the
milk intending to kill their children. They attributed this criminal act to their
social problems.
Inadequate disposal and management of solid wastes and improper
design of sewage network constitute a favorable environment for pests
breeding including rodents in Gaza City. This forced inhabitants to combat
such pests by using different organophosphorus poisons at a large scale.
They usually put the poison in a piece of biscuits, bread or meat. Children are
the most vulnerable victims for such poisons because of their behavior and
playing activities (Tulve et al., 2002). Therefore, eating of poisoned biscuits,
bread or meat was the main cause of poisoning in our target children.
Intensive use and/or misuse of pesticides with poor protective
measures threaten both children and farmers with pesticide poisoning (Yassin
et al., 2002). The poisoned children in the present study as a result of field
and garden sprayed pesticides agreed with the finding of Safi (2000) who
reported several cases of toxicity or death among human exposed to different
types of OP in the Gaza strip. Although a low percentage of the interviewed
children sponsors store pesticides in the home, this practice still puts children
at high risk. Several cases of pesticide poisoning were reported in children
from 2.5 to 6 years old, 30 to 90 minutes after ingestion of an improperly
stored liquid pesticide (Aydin et al., 1997 and Yaramis et al., 2000). Such
practice was also considered to be one of the main problems associated with
pesticide use and its management in developing countries (Wesseling et al.,
1997).
Most of children sponsors reported the use pesticides in their homes to
combat insects. In addition, few of them claimed treatment of pets (especially
cats/dogs) with pesticides and the more importantly is the use of pesticides for
treatment of their children with head lice as reported by some children
sponsors. Pesticides applied inside the home contaminate carpets, sofas,
mattresses, and other household furnishings. This will be a major source of
exposure to children, who ingest particles adhering to food, surfaces in the
home, and the skin, as well as absorption through the skin. Similar studies
reported domestic use of OP and their toxic impact on children ( Zwiener et
al., 1988). In addition, intoxication of children with amitraz pesticide,
commonly used for the control of ticks and mites in dogs, cats, cattle and
sheep, was reported (Caksen et al., 2003). Direct application of pesticide
products to children as in case of lice treatment may cause serious illness and
could be fatal. This may be attributed to the fact that children have more skin
surface for their size which making them more vulnerable to pesticide skin
poisoning than adults. Severe permethrin pesticide toxicity in children due to
inappropriate home treatment for head lice control was reported (Stremski et
al., 2002).
The results presented here showed that the majority of children
sponsors who have gardens or farms spraying them in the presence of their
children and did not store pesticide bottles in a safe place. Spraying activities
in the presence of children making them to be exposed to pesticide drifts. The
ease access of such drifts to children because of more skin surface for their
size and their higher respiratory rate make pesticide poisoning unavoidable.
Brenda et al. (1999) reported that farmers' children can be exposed to OP
through consumption of contaminated food, by household use of pesticides,
as a result of drift from nearby agriculture applications, by contaminated
breast milk from their farm worker mothers, by playing in the fields, and
through pesticides tracked into their homes by their parents or other
household members working in fields. Detectable levels of organophosphate
metabolites were found in farmers' families in five agricultural communities in
rural El Salvador (Azaroff, 1999).The unsafe storage of pesticides reported
here by children sponsors facilitates the accidental ingestion of pesticide
poisoning among children. Similar practice of unsafe storage of pesticides
was previously reported among farmers in the Gaza Strip (Abd Rabou et al.,
2002 and Yassin et al., 2002).
Symptoms associated with OPP among children were diagnosed and
reported once the poisoned child admitted to the hospital. Such symptoms
probably due to inhibition of the enzyme acetylcholinesterase and
accumulation of acetylcholine which acts on its receptors. The diagnosis was
confirmed by measuring the level of serum cholinesterase on admission of the
child to the hospital. The low level of the enzyme assures OPP and the
consequent cholinergic crisis symptoms. The particular clinical features
depends on the type of these receptors and their location i.e. muscarinic,
nicotinic or central nervous system receptors. The most common diagnosed
symptoms reported in our study were pin point pupils, vomiting, drawsy,
conscious and convulsions. Similar data were reported in many countries
including the neighboring ones (Sherman, 1995; Lifshitz et al. 1999; Alarcon
et al., 2005; DeAnda et al., 2009 and El-Naggar et al., 2009).
Researchers have identified 2 types of cholinesterase in human serum-
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). However,
researchers have also found that 1) levels of serum AChE are much lower
than those of serum BuChE (Wilson et al., 1997) making the statistical power
of serum AChE to detect changes from exposure to be reduced and 2) .
serum AChE values are useful for assessing exposures for experimental
animals, such as rats, which have serum AChE levels of 30% to 50%,
whereas human serum AChE levels are much lower (Wilson and Henderson,
1992). Therefore, in the present study serum BuChE (serum cholinesterase)
was used as a biomarker for OPP. The inhibition of serum BuChE has been
taken by scientists as a biomarker for exposure to OP (Hillman, 1994; Misra et
al., 1994; Stefanidou et al., 2003 and Safi et al., 2005). Therefore, it can be
safely mentioned that the inhibition of serum cholinesterase observed in this
study is a result of OPP. Such inhibition was in agreement with other authors
who reported different levels of inhibition in cholinesterase activity among
children exposed to OP (McConnel et al., 1999; Gamlin et al., 2007 and El-
Naggar et al., 2009).
When serum cholinesterase activity was assessed among the most
common diagnosed symptoms in organophosphorus poisoned children, the
lowest enzyme activity was registered in drawsy followed by conscious, pin
point pupils, vomiting and finally in convulsions. Changes in the mean activity
of the enzyme was significantly associated with drawsy, conscious and pin
point whereas no significant association was found in cholinesterase activities
with convulsion and vomiting. This means that the particular clinical symptom
depends on the amount of serum cholinesterase reduction due to OPP i.e. the
greater depression in the enzyme activity, the more severe the poisoning
condition. Experts have recommended the removal of an individual from the
workplace if his serum cholinesterase activity falls below 60% (Environmental
Health Criteria, 1986). In this context drawsy, conscious and pin point
diagnosed symptoms in this study may indicate severe OPP level and need
urgent intervention. Therefore, the amount of serum cholinesterase reduction
due to OPP will be used as a predictor of poisoning severity. Knowing severity
of poisoning could help us to choose the suitable dose of atropine for
treatment and to determine the follow up period of treatment. However, it was
not determined the threshold for severity in this study. Therefore, further
studies are recommended in this regard.
The present data revealed significant increase in serum glucose level
of pesticides the poisoned children compared to the controls. This finding is in
agreement with that reported by Barrueto et al., (2003) in their case report
study of a female child brought to an emergency department at New York city
as a result of playing with rodenticide product. Her initial blood glucose was
108 mg/dl. Balali-Mood and Balali-Mood, (2008) reported that hyperglycemia
is common during OPP and it should be considered and corrected. Elevation
of serum amylase and lipase may reveal acute pancreatities. Also, serum
glucose level was elevated in response to OP administration in experimental
animals (Yassin, 1998). Insecticides may directly or indirectly play a specific
role in pancreatic secretion (Matsumura, 1995), gluconeogenesis process,
and glycogen metabolism or glucose oxidation. When the relationship
between cholinesterase and glucose levels was tested, Pearson's correlation
test showed negative significant correlation (r=0.321, p=0.001). This strong
association meant that elevation in serum glucose accompanied with
depression in serum cholinesterase is more likely attributed to OPP in
children.
Concerning the impact of OP on liver the enzymes, the results showed
that the average levels of serum ALT and AST and ALP in the poisoned
children were significantly higher than those in the controls. Such elevation of
liver enzymes as a result of pesticides exposure including organophosphorus
was documented by other authors (Anwar, 1997; Goel et al., 2000; Altuntus et
al., 2002; Yassin, 2003 and Khan et al., 2008). Liver is the center of
biotransformation and detoxification of foreign compounds and is the most
vulnerable to the chemical assaults (Kulkarni and Hodgson, 1980). Pesticide
exposure causes liver damage and leakage of cytosolic enzymes from
hepatocytes and other body organs into blood (Dewan et al., 2004). Elevation
of liver enzymes may also be due to increased gene expression due to long
term requirement of detoxification of pesticides. When the relationship
between cholinesterase and ALT, AST or ALP levels was tested, Pearson's
correlation test revealed negative correlation between cholinesterase and
ALT, AST or ALP activities (r=0.291, 0.210 and 0.014, respectively). However,
this correlation reached statistically significant level for both ALT and AST
(p=0.003 and 0.032, respectively) but not for ALP (p=0.890). The strong
association between cholinesterase and ALT or AST implied that these two
enzymes are more affected than ALP by OP poisoning in children. Therefore,
one can say that the effect of OP on liver enzymes of concern is in the order
of: ALT>AST>>ALP. This could be true if considered ALT is the most specific
enzyme for liver function.
The influence of OPP on kidney function of the exposed children was
assessed through the measurement of urea and creatinine. Urea and
creatinine levels were increased significantly in the poisoned children
compared to the controls. This is in agreement with that reported in other
studies (Gallo and Lawryk, 1991; Yousaf et al., 2003; Attia, 2006 and Khan et
al., 2008). In addition, nephrotoxic effect of OP have been reported
(Kossmann et al., 1997 and Poovala et al., 1999). Urea is formed by the liver
as an end product of protein breakdown and is one marker of the kidney
function (Debra Manzella, 2008). Increase in serum urea observed here may
be due to 1) impairment in its synthesis as a result of impaired hepatic
function as mentioned above, 2) disturbance in protein metabolism and 3)
decrease in the filtration rate of the kidney. Creatinine is a waste product that
is normally filtered from the blood and excreted with the urine. Increase in
creatinine levels in response to OPP are indicating renal diseases. However,
this change may be not significant (Parron et al., 1996 and Yassin, 2003).
Consequently, it is difficult to determine the onset of such changes and this
may lead to controversial results. Therefore, it must watch the creatinine
levels carefully to determine how much function the kidneys have and this
does vary slightly. Again Pearson's correlation test revealed negative
correlation between cholinesterase and urea or creatinine levels (r=0.079 and
0.212, respectively). This correlation was significant for creatinine (p=0.030)
and not significant for urea (p=0.426). The strong association between
cholinesterase and creatinine implies that creatinine is acually affected by OP
poisoning in children. Therefore, creatinine is a better indicator for kidney
function even in the case of OP exposure.
As indicated in this results, serum potassium and phosphorus
concentrations were decreased significantly in the cases compared to the
controls whereas sodium and calcium showed non significant increase. The
amount of change in serum potassium and phosphorus associated with OPP
was small, but significant. This is because change in body electrolytes is
generally under critical control by different body mechanisms. Therefore, the
statistical power of serum potassium and phosphorus to detect changes from
OP exposure is reduced. Balali-Mood and Balali-Mood, (2008) reported that
acid-base and electrolyte disturbances are common during OPP.
Hypokalemia is common and should be considered and corrected. The
significant decrease in phosphorus concentration could be also attributed to
impaired kidney function in response to pesticides exposure. It seems that OP
had little effect on sodium and calcium implying that their transport systems
and metabolism did not affect much. When the relationship between
cholinesterase and phosphorus was tested, Pearson's correlation test showed
positive significant correlation (r=0.234, p=0.017). This strong association do
confirm the above idea that the decrease in serum phosphorus accompanied
with the depression in serum cholinesterase is more likely attributed to the
effect of OP on the kidney function in children.
Concerning protein profile, the results demonstrated significant
decreases in the averages of total protein, albumin and globulin
concentrations in the cases compared to the controls. These findings are in
agreement with that observed in other studies as a result of OP exposure
(Jyotsna et al., 2003 and Balali-Mood and Balali-Mood, 2008). It is tempting to
speculate that decreased serum total protein may be due to lowered synthesis
of albumin in liver and a rise in globulins (mostly γ-globulins) which consumed
in production of antibodies due to children exposure of OP pesticides.
Pearson's correlation test showed positive non significant correlation between
cholinesterase and total protein. This means that the decrease in total protein
observed in OP poisoned children was not totally depend on the account of
the depression of cholinesterase, but instead it depends mainly on albumin
and globulin decreases as discussed above.
Complete blood count was performed for every poisoned child admitted
to the hospital. Total white blood cell count was markedly elevated in the
cases compared to controls. For differential white blood cells, lymphocytes
were also higher in the cases whereas neutrophils were lower. Like white
blood cells, blood platelets were significantly increased in the cases. Similar
results were addressed as a result of exposure to OP in adults and children
(Safi et al., 2005 and Rastogi et al., 2008). In addition, pesticides including OP
insecticides have been shown to have hematotoxic properties and may cause
agranulocytosis and neutropenia (Parent-Massin and Thouvenot, 1993). The
induction of white blood cell count indicates the activation of a defense
mechanism and the immune system, which could be a positive response for
survival (Wesseling et al., 1997). Leukocytosis has been recorded following
the administration of insecticides, including organophosphates (Brown et al.,
1990 and Sungur and Guven, 2001).
Regarding primary blood indices, red blood cell count was not
significantly changed between the cases and the controls. However,
hemoglobin and hematocrit were significantly decreased in the cases
compared to the controls. Secondary blood indices including MCV, MCH and
MCHC were also found to be lower in the cases. These results are consistent
with those of other studies dealing with OP exposure in humans (Ray, 1992;
Jyotsna et al., 2003 and Leilanie and Prado-Lu, 2007). The observed
decrease in hemoglobin content and hematocrit value in children could be
attributed to the decreased size of red blood cells or the impaired biosynthesis
of heme in bone marrow as a result of pesticide exposure (Isselbacher et al.,
1992 and Zayed et al., 1993). The decrease in the secondary blood indices is
more likely to be a consequence of the decreased observed in the primary
indices.
On the light of the results, it is acceptable to say that the common
causes of OPP in the Gaza Strip include domestic use and unsafe storage of
pesticides, and spraying pesticides in the presence of children. Serum
cholinesterase still proven to be the best biomarker for OPP. The particular
clinical symptom depends on the amount of serum choliesterase reduction i.e.
the greater depression in the enzyme activity, the more severe the poisoning
condition. Liver, kidney and blood of children were affected by OPP putting
their lives in a real threat.
Chapter 6 Conclusions and recommendations
6.1 Conclusions 1. The major causes of OPP in children in the Gaza Strip were domestic use
of pesticides particularly to fight rodents and unsafe storage of pesticides, and
spraying pesticides in their presence.
2. The most common diagnosed symptoms of OPP in children were pin point
pupils followed by vomiting, drawsy, conscious and then by convulsions.
3. There was a depression in serum cholinesterase in the cases compared to
the controls. Changes in enzyme activity was significantly associated with
drawsy, conciuos and pin piot.
4. Glucose level was significantly increase in the patients compared to the
controls.
5. Liver enzymes ALT, AST and ALP were significantly higher in the cases
6. Urea and creatinine levels were increased in the cases compared to the
controls.
7. Of the electrolytes studied, potassium and phosphorus were decreased
significantly in the cases.
8. There were significant decreases in total protein, albumin and globulin in
the cases compared to the controls.
9. White blood cell count and blood platelets were higher in the cases.
10. Hemoglobin and hematocrit, MCV and MCH were significantly decreased
in the cases compared to the controls.
11. Pearson's correlation test showed negative significant correlation between
cholinesterase and glucose, ALT, AST or creatinine. On the other hand,
positive significant correlation between cholinesterase and phosphorus was
achieved.
6.2 Recommendations 1. Restriction the use of pesticides in home as can as possible
2. Not allowing ease access of children to reach poison intended to kill
rodents.
3. Use of pesticides for treatment of children with head lice is forbidden
4. Avoidance of spraying pesticides in the presence of children
5. Storage of pesticide bottles in a safe place
6. Serum cholinesterase as a biomarker for monitoring OPP is highly
recommended.
7. Serum glucose, ALT, AST and creatinine could be used as primary
indicators for OPP.
8. Enhancement of people awareness towards the safe use of pesticides and
their poisoning hazards on children by launching educational programs and
workshops.
9. Further studies are needed to clarify the threshold for severity of OPP
which could be useful to choose the suitable dose of atropine for treatment.
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Annex 1
Annex 2
Annex 3
Annex 4
Questionnaire: OPP in children
Name of child……………………………. Age …………………..
Patient number…………………………………
Date of admission to hospital………………………….
1. Has your child eaten any of the following in the last 12 hours
Freshly sprayed fruit Yes No
Freshly sprayed vegetables Yes No
Poisoned biscuits, bread or meat Yes No
2. Has your child drink any of the following in the last 12 hours
Tap water from empty bottles Yes No
Pesticide stored in bottles Yes No
Deliberate pesticide poisoned milk Yes No
Unknown cause of pesticides poisoning among children?
Yes No
3. Do you have any pets (esp. cats/dogs)? Yes No
4. Has anyone in your household recently had treatment for headlice?
Yes No
5.Have you recently used any domestic insecticide sprays, solution, fly-
strips, plug- ins or pellets in your home or garden? Yes No
Questions for father and mother
6.Do you have a garden or a farm? Yes No
7. Do you spray your garden or farm in the presence of your children?
Yes No
8.During spray are your children:
-Drinking Yes No
-Eating Yes No
-playing Yes No
9.Where do you store pesticides bootless or cans ?
-In specific storage in safe site Yes No
-Other places, please specify……………………….?
10. what are you doing with the empty bootless or cans.?
-for storage water Yes No
-for storage food Yes No
-for others Yes No
11.Unknown cause of pesticides poisoning among children?
Yes No