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