Development of a Biosensor for Methyl Parathion...

i

Development of a Biosensor for Methyl Parathion

Detection and Genetic Risk Assessment of

Exposed Population

By

Muhammad Idrees

CIIT/FA07-PBS-004/ISB

PhD Thesis

in

Biosciences

COMSATS Institute of Information Technology

Islamabad- Pakistan

Spring, 2015

ii

COMSATS Institute of Information Technology


Detection and Genetic Risk Assessment of Exposed

Population

A Thesis Presented to

COMSATS Institute of Information Technology, Islamabad

in partial fulfillment

of the requirement for the degree of

PhD (Biosciences)

By

Muhammad Idrees


Spring, 2015

iii



Population

A Post Graduate Thesis submitted to the Department of Biosciences as partial

fulfillment of the requirement for the award of the Degree of PhD (Biosciences).

Name Registration Number

Muhammad Idrees CIIT/FA07-PBS-004/ISB

Supervisor

Dr. Syed Habib Ali Bokhari

Professor

Department of Biosciences

COMSATS Institute of Information Technology (CIIT)

Islamabad Campus

iv

Final Approval

This Thesis titled



Population

by

Muhammad Idrees


Has been approved

For the COMSATS Institute of Information Technology, Islamabad

External Examiner 1: __________________________________________

External Examiner 2: __________________________________________

Supervisor: ______________________________________________


Professor & Chairman, Dept of Biosciences, CIIT Islamabad

HoD: __________________________________________________

Dr. Raheel Qamar (T.I.)

Professor, Dept of Biosciences, CIIT Islamabad

Chairman:


Professor, Dept of Biosciences, CIIT Islamabad

Dean, Faculty of Science: ___________________________________

Dr. Arshad Saleem Bhatti (T.I.)

Professor, Dept of Physics, CIIT Islamabad

v

Declaration

I Muhammad Idrees, CIIT/FA07-PBS-004/ISB hereby declare that I have produced the

work presented in this thesis, during the scheduled period of study. I also declare that I

have not taken any material from any source except referred and the amount of plagiarism

is within acceptable range. If a violation of HEC rules on research has occurred in this

thesis, I shall be liable to punishable action under the plagiarism rules of the HEC.

Date: ______________________ Signature of the Student:

_____________________

Muhammad Idrees


vi

Certificate

It is certified that Muhammad Idrees, CIIT/FA07-PBS-004/ISB has carried out all the work

related to this thesis under my supervision at the Department of Biosciences, COMSATS

Institute of Information Technology, Islamabad and the work fulfills the requirement for

award of PhD degree.

Date: ___________________

Supervisor:

________________________


Professor& Chairman

Department of Biosciences

Head of Department:

________________________

Dr. Raheel Qamar

Professor

Head, Department of Biosciences, CIIT, Islamabad

vii

DEDICATION

To Almighty ALLAH and the Holy Prophet

Muhammad (P.B.U.H)

&

My Teachers, Parents & Family

ACKNOWLEDGMENTS

viii

Millionth gratitude to ALLAH ALMIGHTY, The Most Beneficent and The Most Merciful who

is my creator and my most well-wisher, who bestowed me with the potential to seek knowledge

and to explore some of the many aspects of His creation. Countless blessings and clemencies of

Allah upon our HOLY PROPHET MUHAMMAD,Sallallaho-alaihe-wa-alehi-wa-barik-wa-

sallim, and His Ahl-e-Bait and Ashaab, who are the fortune of knowledge, who took the humanity

out of the abyss of ignorance and elevated it to the zenith of consciousness.

It would have not been possible to write this thesis without the help and support of the kind people

around me, to only some of whom it is possible to give particular mention here. First of all I wish

to express my heartiest and sincere gratitude to my Supervisor Prof. Dr. Habib Ali Bokhari for

his invaluable guidance, suggestions, and constant encouragement. His constant support and

personal guidance has provided a good basis for the present thesis. I have learnt a lot from him, not

only the science and research but also art of life. I am thankful to him for his kind support.

I wish to express my warm and sincere thanks to Prof. Dr. Jayaraman Sivaraman (NUS,

Singapore) for giving me opportunity to work in his labs under his personal supervision and

guidance. In addition I am also very grateful to Prof. K. Swaminathan (NUS, Singapore) for

providing me all the help and lab facilities for some essential parts of this thesis. I am also very

thankful to Prof. Dr. Raheel Qamar (T.I) (COMSATS, Islamabad) for all the help, guidance and

support during my course work and research work.

In addition some of the faculty members have been very kind to me and they always inspired and

helped me, whenever I needed their help. Dr. Ali Mustajab, Dr. Rani Faryal, Dr. Faraz Malik, Dr.

Jobi (NUS, Singapore) are amongst those sincere seniors who helped and encouraged me to

accomplish my tasks in research.

I feel pride to acknowledge the endless efforts of my family (Mother, Father, brothers and sisters

and wife). They are amongst the one of the biggest blessings of Allah on me. May Allah always

bless them with His blessings! May Allah always bless any one who has done anything good for

me.

It was really a great honor for me to have studied and worked under the guidance of the best

faculty of its kind, which includes Dr. Raheel Qamer, Dr. Habib Bokhari, Dr. Kayani, Dr.

Shahid Chohan, Dr. Rani Faryal and Dr. Ansar. They have nourished my mind and soul and I

have obtained a lot of understanding of the new advancements in the fields of Biological and

Life sciences from them. Each of them is a legend in his / her field of work.

ix

Some of my friends are very special to me due to their sincerity. Dr. Thangavelu, Dr. Manjeet,

Dr. Verindra Kumar, Dr. Imran, Miss Yasmin and Dr. Shazia are amongst them. I can never

forget their sincere efforts for me. Each of them has contributed a lot by one or the other way

for the accomplishment of this research work. I am also thankful to Miss Maimoona for helping

me in formatting of the front pages. Last but not the least I would like to thank Higher Education

Commission (HEC) of Pakistan for providing me Indigenous Scholarship for my PhD studies

and IRSIP scholarship for my research at NUS, Singapore. HEC and COMSATS (CIIT,

Islamabad) provided funds for the project to which my work is a small effort.

`

Muhammad Idrees


`

ABSTRACT

Methyl parathion is one of the highly toxic organophosphate pesticides which contaminates

x

the environment and exerts toxic effects in the non-targeted living organisms. Its detection and

removal from the environment is needed in order to protect both the public and the environment

from its hazardous effects. We studied risk assessment to pesticide toxicity (based on case studies),

detection of methyl parathion (by using purified green fluorescent protein as a biosensing molecule)

and biodegradation of methyl parathion (by indigenous bacterial isolates immobilized in sol gel

matrices).

Paraoxonase is a protein with enzymatic function which hydrolyzes toxic metabolites of

methyl parathion and other organophosphate pesticides. We determined serum paraoxonase and

serum arylesterase activities alongwith genotypes of respective groups recruited for the study.

Various groups of the study population included healthy non-pesticide exposed male Punjabis,

pesticide users, familial hypercholesterolaemia patients, glaucoma patients and appropriate

controls. The results of the healthy population provided a baseline data (normal values). Study on

pesticide users indicated significant differences of PON1 genotypes and phenotypes between the

healthy pesticide users and the pesticide users who developed toxic effects of pesticides during

spraying season. Serum paraoxonase and arylesterase activities were significantly low in

hypercholesterolemia patients as compared to controls. There was no significant difference of

PON1 genotype or phenotypes between glaucoma patients and age and gender matched controls.

However age dependant decrease in serum paraoxonase and arylesterase activities explained the

role of PON1 in glaucoma. The results of these studies will be helpful in understanding preventive

measures to improve the status of public health.

Detection of methyl parathion is relatively costly with available methods and low cost and

easy to use biosensor systems are needed for large scale screening of agricultural areas and other

environmental segments. Thus we studied methyl parathion detection based on a simple method of

fluorescence quenching. The linear decrease in the fluorescence of green fluorescent protein with

increasing concentration of methyl parathion showed that fluorescence quenching based method

can be utilized for low cost detection of methyl parathion. In addition, a novel fluorescent protein

(HriGFP) was characterized for its physico-chemical properties to provide a baseline for future

studies.

Bacteria play a major role in degrading pesticides in the environment. Bacterial enzymes

(esterases) convert the pesticides into metabolites which can be used as nutrients by the bacteria

for their growth and proliferation. We isolated bacterial strains from agricultural areas

contaminated with methyl parathion and other pesticides and characterized them for their combined

xi

potential of antimicrobial and heavy metal resistance and utilizing methyl parathion as a source of

nutrients. In addition, a short listed consortium of the bacterial strains was immobilized in sol-gel

and their survival in sol gel was studied. The results indicate that these environmental bacteria are

capable of utilizing methyl parathion as a source of nutrient and sol gel immobilization can be

helpful in co-survival of different bacterial strains for their application in bioremediation.

On the whole, this thesis describes PON1 genotype and phenotype patterns in Pakistani

populations, use of GFP as a biosensor molecule for detection of methyl parathion and isolation

and characterization of bacterial strains for methyl parathion degrading activities. The study not

only gives insight about the risk assessment and ultimately prevention of toxic effects of methyl

parathion but also describes a proof of principle based on simple biotechnology approach for its

detection. The results of this thesis will be a valuable source of information for public health

managers, clinicians, policy makers, agricultural workers and bio-medical researchers for ultimate

improvement of health status of general population as well as pesticide users.

TABLE OF CONTENTS

1.0 General Introduction and Literature review……………………….......………… 1

xii

1.1 Methyl parathion and Organophosphate Pesticides--------------------------------------1

1.1.1 Methyl parathion---------------------------------------------------------------------1

1.1.2 Pharmacokinetics of methyl parathion-------------------------------------------2

1.1.3 Methyl parathion toxicity revealed by animal models--------------------------2

1.1.4 Effects of environmental contamination with methyl parathion--------------5

1.1.5 Methyl parathion toxicity in humans---------------------------------------------6

1.1.6 Current analytical techniques for methyl parathion detection----------------8

1.2 Paraoxonase 1 (PON1)---------------------------------------------------------------------8

1.2.1 PON1 gene Polymorphisms-------------------------------------------------------9

1.2.1.1 Q/R polymorphism in coding region (position 192)--------------------9

1.2.1.2 PON1 L/M polymorphism-----------------------------------------------9

1.2.2 Physiological roles of PON1 protein------------------------------------------11

1.3 Problem statement------------------------------------------------------------------------------13

1.4 Objectives of the study------------------------------------------------------------------------13

2.0 Paraoxonase-1 Gene Polymorphism in Punjabi Population of Pakistan----------14

2.1. Introduction-------------------------------------------------------------------------------------14

2.2. Materials and methods------------------------------------------------------------------------16

2.2.1 Study Population--------------------------------------------------------------------16

2.2.2 Samples-------------------------------------------------------------------------------16

2.2.3 Determination of PON1 SNPs-----------------------------------------------------16

2.2.4 PON1 paraoxonase activity measurement --------------------------------------18

xiii

2.2.5 PON1 arylesterase activity measurement ---------------------------------------19

2.2.6 Statistical analysis-------------------------------------------------------------------19

2.3. Results ------------------------------------------------------------------------------------------20

2.3.1 Demographic properties----------------------------------------------------------20

2.3.2 PCR-RFLP-------------------------------------------------------------------------20

2.3.3 Allele and genotype frequencies------------------------------------------------24

2.3.4 Frequencies of combined genotypes-------------------------------------------24

2.3.5 Serum paraoxonase activities according to PON1 L55M and PON1

Q192R-----------------------------------------------------------------------------26

2.3.6 Serum arylesterase activities according to PON1 L55M and PON1 Q192R-

---------------------------------------------------------------------------27

2.3.7. Combined results of paraoxonase and arylesterase activities according to

PON1 L55M and PON1 Q192R-------------------------------------------------------28

2.3.8. PON1 Enzyme activities with respect of combined genotypes------------29

2.4. Discussion--------------------------------------------------------------------------------------30

2.5. Conclusions------------------------------------------------------------------------------------37

3.0 (Case study-2): Paraoxonase-1 Genotypes and Enzyme Activities in Pakistani

(Punjabi) Pesticide Sprayers: Identification of Population Susceptible to Risk of

Pesticide Toxicity----------------------------------------------------------------------------------38

3.1. Introduction-----------------------------------------------------------------------------------38

3.2. Materials and methods----------------------------------------------------------------------40

3.2.1. Study Population-------------------------------------------------------------------40

3.2.2. Methods -----------------------------------------------------------------------------40

xiv

3.3. Results------------------------------------------------------------------------------------------41

3.3.1. Study population-------------------------------------------------------------------41

3.3.2. Types of pesticides used-----------------------------------------------------------42

3.3.3. Personal Protective Equipments (PPEs)-----------------------------------------43

3.3.4. Safety Precautions about wind direction----------------------------------------44

3.3.5. Safety precautions after working with pesticides------------------------------45

3.3.6. Sources of training and guidance-------------------------------------------------46

3.3.7. Direct body contact with pesticides during work------------------------------47

3.3.8. Signs and symptoms of acute toxicity recorded in affected individuals----48

3.3.9. PON1 SNPs-------------------------------------------------------------------------49

3.3.10. Serum arylesterase activities according to PON1 L55M and PON1 Q192R-

------------------------------------------------------------------------------53

3.3.11. Serum paraoxonase activities according to PON1 L55M and PON1

Q192R--------------------------------------------------------------------------------------54

3.3.12. Serum arylesterase activities according to combined genotypes----------55

3.3.13. Serum paraoxonase activities according to combined genotypes --------56

3.4. Discussion: ------------------------------------------------------------------------------------57

3.5. Conclusions:-----------------------------------------------------------------------------------64

4.0 Serum Paraoxonase and Arylesterase Activity in Familial Hypercholesterolemia

Patients with Mutated LDLR Gene------------------------------------------------------------65

4.1. Introduction-----------------------------------------------------------------------------------65

4.2. Patients and Methods-----------------------------------------------------------------------67

xv

4.2.1 Subjects-------------------------------------------------------------------------------67

4.2.2. Methods -----------------------------------------------------------------------------67

4.3. Results------------------------------------------------------------------------------------------68

4.3.1. Demographic characteristics-----------------------------------------------------68

4.3.2. Pedigree and PCR-RFLP results -----------------------------------------------69

4.3.3. Prevalence of PON1 genotypes-------------------------------------------------71

4.3.4. Serum arylesterase activities according to PON1 L55M and PON1 Q192R

--------------------------------------------------------------------------------------------------75

4.3.5. Serum paraoxonase activities according to PON1 L55M and PON1 Q192R-

----------------------------------------------------------------------------------------76

4.3.6. Serum arylesterase activities according to combined genotypes-----------77

4.3.7. Serum paraoxonase activities according to combined genotypes ----------78

4.4. Discussions-------------------------------------------------------------------------------------79

4.5. Conclusions------------------------------------------------------------------------------------83

5.0. (Case study-4): Serum Paraoxonase and Arylesterase Activity in Primary

Glaucoma Patients --------------------------------------------------------------------------------84

5.1. Introduction-----------------------------------------------------------------------------------84

5.1.1. Normal eye structure and flow of Aqueous humor----------------------------86

5.1.2. Difference between normal eye and glaucoma---------------------------------87

5.1.3. Difference between OAG and CAG---------------------------------------------88

5.1.4. Optic nerve damage due to glaucoma ----------------------------------------- 89

5.1.5. Altered visual field due to glaucoma--------------------------------------------90

xvi

5.2. Patients and Methods-----------------------------------------------------------------------91

5.2.1. Subjects:-----------------------------------------------------------------------------91

5.2.2. Methods:-----------------------------------------------------------------------------93

5.3. Results:-----------------------------------------------------------------------------------------94

5.3.1. Comparison of PON1 genotypes distribution----------------------------------96


------------------------------------------------------------------------------------------------99


---------------------------------------------------------------------------------------------100

5.3.4. Serum arylesterase activities according to combined genotypes-----------101

5.3.5. Serum paraoxonase activities according to combined genotypes----------102

5.4. Discussions:----------------------------------------------------------------------------------103

5.5. Conclusions:---------------------------------------------------------------------------------106

6.0 Detection of Methyl Parathion by Using Purified Green Fluorescent Protein

(GFPuv) as a Biosensor Molecule-------------------------------------------------------------107

6.1 Introduction---------------------------------------------------------------------------------107

6.2. Materials and Methods--------------------------------------------------------------------108

6.2.1. Selection of Fluorescent protein -----------------------------------------------108

6.2.2. Expression and purification of GFPuv----------------------------------------108

6.2.3. Fluorescence assay--------------------------------------------------------------109

6.3. Results:----------------------------------------------------------------------------------------110

6.3.1. Expression of GFPuv ------------------------------------------------------------110

xvii

6.3.2. IMAC purification of GFPuv ---------------------------------------------------111

6.3.3 FPLC -------------------------------------------------------------------------------112

6.3.4 Fluorescence quenching assay --------------------------------------------------113

6.4 Discussions -----------------------------------------------------------------------------------114

6.5 Conclusions ----------------------------------------------------------------------------------118

7.0 Characterization of a Novel Fluorescent Protein from Hydnophora rigida with

Green Emission ---------------------------------------------------------------------------119

7.1. Introduction --------------------------------------------------------------------------------119

7.2. Materials and Methods -------------------------------------------------------------------120

7.2.1 HriGFP expression and purification ---------------------------------------120

7.2.2 Dynamic light scattering (DLS) ------------------------------------------------120

7.2.3 Peptide Mass Finger print (PMF) -----------------------------------------------121

7.2.4 Searching for similar proteins ---------------------------------------------------121

7.2.5 Identification of fluorophore ----------------------------------------------------121

7.2.6 Structure Prediction on the basis of similar templates -----------------------122

7.3. Results ----------------------------------------------------------------------------------------125

7.3.1 HriGFP expression and purification --------------------------------------------125

7.3.2 The Dynamic Light Scattering (DLS)------------------------------------------126

7.3.3 The peptide mass fingerprint ----------------------------------------------------126

7.3.4. Sequence analysis Results ------------------------------------------------------127

7.3.5. Phylogenetic tree -----------------------------------------------------------------128

7.3.6. Homology modeling: prediction of 3D structure ---------------------------131

xviii

7.3.7. Fluorophore identification -----------------------------------------------------132

7.4. Discussions ----------------------------------------------------------------------------------133

7.5. Conclusions ---------------------------------------------------------------------------------137

8.0. Methyl Parathion Biodegradation: Isolation and Characterization of Methyl

Parathion Utilizing Bacterial Isolates for Bioremediation Applications ------------138

8.1. Introduction---------------------------------------------------------------------------------138

8.2. Materials and methods--------------------------------------------------------------------139

8.2.1. Soil Samples for bacterial isolation -------------------------------------------139

8.2.2. Isolation of Methyl parathion utilizing bacteria -----------------------------142

8.2.3. Selection of pure bacterial colonies -------------------------------------------142

8.2.4. Identification of isolates --------------------------------------------------------143

8.2.5. Methyl parathion Hydrolase assay --------------------------------------------143

8.2.6. Heavy Metal tolerance ----------------------------------------------------------144

8.2.7. Antibiotic resistance ----------------------------------------------------------144

8.2.8. PCR for integrons and antimicrobial resistance genes ----------------------145

8.3. Results ----------------------------------------------------------------------------------------148

8.3.1 Methyl parathion utilizing bacterial isolates ----------------------------------148

8.3.2. Heavy Metal tolerance ----------------------------------------------------------150

8.3.3. Antibiotic resistance -------------------------------------------------------------151

8.3.4. Antimicrobial resistance genes and integrons --------------------------------152

8.4. Discussions ----------------------------------------------------------------------------------154

xix

8. 5. Conclusions ---------------------------------------------------------------------------------160

9.0 Methyl Parathion Bioremediation (Sol Gel Immobilization of a Consortium of

Methyl Parathion Utilizing Bacterial Isolates) -------------------------------------------161

9.1. Introduction --------------------------------------------------------------------------------161

9.2. Material and methods --------------------------------------------------------------------162

9.2.1. Consortium of Bacterial Isolates ----------------------------------------------162

9.2.2. Methyl parathion hydrolase activity of Consortium ------------------------162

9.2.3. Growth pattern of Consortium members -------------------------------------162

9.2.4. Cell immobilization and stability in sol-gel ----------------------------------162

9.2.4.1. Cell immobilization and stability in TEOS derived sol-gel -----163

9.2.4.2. Cell immobilization and stability in sodium silicate derived sol-gel-

-----------------------------------------------------------------------164

9.3. Results ----------------------------------------------------------------------------------------165

9.3.1. Selection of Bacterial Consortium for sol gel studies:-----------------------165

9.3.2. Methyl parathion degrading enzymatic activity of short listed consortium--

-------------------------------------------------------------------------------------166

9.3.3. Growth pattern of bacterial strains of the consortium-----------------------167

9.3.4. Cell immobilization and stability in sol-gel-----------------------------------168

9.3.4.1 Bacterial survival in TEOS based sol-gel--------------------169

9.3.4.2 Bacterial survival in sodium silicate based sol-gel----------170

9.3.5. GFP expression in sol-gel immobilized cells --------------------------------171

9.4. Discussions ----------------------------------------------------------------------------------173

xx

9.5. Conclusions ---------------------------------------------------------------------------------176

10.0. References ---------------------------------------------------------------------------------177

11.0 Appendices ---------------------------------------------------------------------------------217

11.1. General Performa for PON1 Studies Participants -------------------------------------224

11.2. Additional Information of PON1 Studies Participants -------------------------------226

12.0 List of Published Research work-------------------------------------------------------231

xxi

LIST OF FIGURES

Fig. 1.1: General structure of OP compounds----------------------------------------------------------1

Fig. 1.2: Different routes of exposure to methyl parathion and other Organophosphate

pesticides----------------------------------------------------------------------------------------------------3

Fig. 1.3: PON1 gene structure -------------------------------------------------------------.------------10

Fig 1.4: Structure of PON1 protein. ------------------------------------------------------------------10

Fig. 1.5: Some important physiological effects of PON1 protein on major organs-----------12

Fig. 2.1: PCR amplified products of PON1 Q192R ------------------------------------------------20

Fig. 2.2: PCR-RFLP results of PON1 Q192R. ------------------------------------------------------21

Fig. 2.3: PCR amplified product of PON1 L55M --------------------------------------------------22

Fig. 2.4: PCR-RFLP results of PON1 L55M. ------------------------------------------------------23

Fig. 2.5: A 3D chart showing pattern of relative frequencies (%) of combined genotypes of

PON1 L55M and PON1 Q192R genotypes in healthy male Punjabis (n = 140) with no history

of exposure to pesticides.------------------------------------------------------------.---------25

Fig. 2.6: Serum paraoxonase activity (U/L) of Pakistani Punjabi healthy male individuals

(with no exposure to pesticides, n = 140) with respect to PON1 L55M and PON1 Q192R

genotypes.----------------------------------------------------------------------------------------------26

Fig. 2.7: Serum arylesterase activity (U/mL) of Pakistani Punjabi healthy male individuals

(with no history of exposure to pesticides) (n = 140) with respect to PON1 L55M and PON1

Q192R genotypes.---------------------------------------------------------------------------27

Fig. 2.8: Serum paraoxonase and arylesterase activities of healthy male Punjabis according

to presence of different genotypes (PON1 Q192R polymorphism: QQ, QR, RR and PON1

L55M polymorphism: LL, LM, MM) of coding region polymorphisms of PON1 gene. -----

---------------------------------------------------------------------------------------------------------28

xxii

Fig. 2.9: Serum paraoxonase (U/L) and arylesterase (U/mL) activities in healthy male

Punjabis with no history of exposure to pesticides (n = 140) according to combined

genotypes of coding region polymorphisms of PON1 gene. ----------------------------------29

Fig. 3.1: Comparison of pesticide usage in studied Pakistani Punjabi Pesticide workers.---

---------------------------------------------------------------------------------------------------------42

Fig. 3.2: Comparison of personal protective equipment (PPE) usage in studied Pakistani

Punjabi Pesticide workers (back pack sprayers).--------------------------------------------- -43

Fig. 3.3: Comparison of safety measure about wind direction in studied Pakistani Punjabi

Pesticide workers (back pack sprayers).------------------------------------------------------- 44

Fig. 3.4: Comparison of safety measures (after pesticide spraying) in studied Pakistani

Punjabi Pesticide workers (back pack sprayers). ---------------------------------------------45

Fig. 3.5: Comparison of sources of training in Pakistani Punjabi Pesticide workers (back

pack sprayers). -------------------------------------------------------------------------------------46

Fig. 3.6: Comparison of direct body contact (direct exposure) with pesticides in studied

Pakistani Punjabi Pesticide workers (back pack sprayers).-----------------------------------47

Fig. 3.7: Signs and symptoms of acute toxic effects of organophosphate pesticides in

affected pesticide workers (n = 101)------------------------------------------------------------ 48

Fig. 3.8: Comparison of genotypes between affected (n = 101) and non affected (n = 100)

spray workers and healthy Punjabis (n = 140).------------------------------------------------ 52

Fig. 3.9: Differences of serum arylesterase activities in affected pesticide sprayers (n =

101) compared with non-affected pesticide workers (n = 100) and healthy non pesticide

exposed Punjabi population (n = 140).-----------------------------------------------------------53

Fig. 3.10: Differences of serum paraoxonase activities (U/L) in affected pesticide

sprayers (n = 101) compared with non-affected pesticide workers (n = 100) and healthy

non pesticide exposed Punjabi population (n = 140) -----------------------------54

xxiii

Fig. 3.11: Comparison of serum arylesterase activity (U/mL) in different genotypes of

affected pesticide sprayers (n = 101) compared with non-affected sprayers (n = 100) and

non- exposed healthy Punjabi population (n = 140) ----------------------------------55

Fig. 3.12: Differences of serum paraoxonase activities (U/L) in affected pesticide

sprayers (n = 101) compared with non-affected pesticide workers (n = 100) and healthy

non pesticide exposed Punjabi population (n = 140) with respect of combined genotypes

of PON1 Q192R and PON1 L55M ----------------------------------------------56

Fig. 4.1: Pedigree of hypercholesterolemia family ----------------------------------------70

Fig. 4.2: Comparison of combined genotype frequencies between healthy Punjabi

population, normal healthy controls and hypercholesterolaemia patients -----------------74

Fig. 4.3: Comparison of arylesterase activity between cases (n = 10) and healthy controls

of family (n = 24) and healthy Punjabi population (n = 140) with respect of QQ, QR, RR,

LL, LM, MM genotypes. -------------------------------------------------------------------------75

Fig. 4.4: Comparison of paraoxonase activity (U/L) between cases (n = 10) and healthy

controls of family (n = 24) and healthy Punjabi population (n = 140) with respect of QQ,

QR, RR, LL, LM, MM genotypes.--------------------------------------------------------------76


of family (n = 24) and healthy Punjabi population (n = 140) with respect of combined

genotypes. ------------------------------------------------------------------------------------------77

Fig. 4.6 Comparison of Paraoxonase activity between cases (n = 10) and healthy controls


genotypes. -----------------------------------------------------------------------------78

Fig. 4.7: Schematic diagram showing normal LDLR gene --------------------------------80

Fig. 5.1: Diagram showing normal structure of eye with normal fluid flow which leads to

normal IOP (Intra Ocular Pressure) for normal eye function --------------------------------86

xxiv







Fig. 5.5: Diagram showing damage to optic nerve with advancement of glaucoma -----89

Fig. 5.6: Diagram showing Tunneling vision (loss of peripheral vision) with advancement

of glaucoma -----------------------------------------------------------------------------------------90

Fig. 5.7: Determination of c/d (cup/disc ratio) of optic nerve head (ONH) (Normal ONH is

shown in this figure) --------------------------------------------------------------------------------92

Fig. 5.8: Comparison of genotypes between Glaucoma patients (PCAG and POAG) with

normal controls and healthy male Punjabi population -----------------------------------------98

Fig. 5.9: Comparison of serum arylesterase activities (U/mL) of Glaucoma patients (PCAG

and POAG) with normal controls and healthy male Punjabi population according to

genotypes. ---------------------------------------------------------------------------------------------99

Fig. 5.10: Comparison of serum paraoxonase activities (U/L) of Glaucoma patients (PCAG

and POAG) with normal controls and healthy male Punjabi population according to genotypes.

---------------------------------------------------------------------------------------------100



combined genotypes. ---------------------------------------------------------------------------101



combined genotypes -------------------------------------------------------------------------102

xxv

Fig 6.1: Expression of GFPuv in E. coli DH5 α transformed with pGFPuv visualized under

UV light ------------------------------------------------------------------------------------110

Fig 6.2: IMAC: Nickel affinity chromatography without adding His tag in GFPuv protein.--

-------------------------------------------------------------------------------------------------------111

Fig 6.3: Purification of GFPuv. Fractions from the center of major peak were collected and

observed for presence of fluorescence.----------------------------------------------------112

Fig. 6.4: Fluorescence quenching effects of methyl parathion (0-200 μM) on purified two

different concentrations (100 nM and 50 nM) of GFPuv protein indicating a biosensing

effect.-----------------------------------------------------------------------------------------------113

Fig 6.5: Protein sequence and Ribbon diagram of the backbone of GFPuv--------------117

Fig. 7.1: HriGFP purification -------------------------------------------------------------------125

Fig. 7.2: Gel filtration chromatography of HriGFP. Elution profile shows that HriGFP

elutes as the monomer. --------------------------------------------------------------------------126

Fig. 7.3: Mass spectrum of tryptic peptides digest of HriGFP ----------------------------127

Fig. 7.4: Sequence similarity of HriGFP with other proteins. Similar residues are shown in

red letters. Accession numbers of respective proteins are given in blue letters on left hand

side.----------------------------------------------------------------------------------------- 128

Fig. 7.5: Neighbor joining tree of Anthozoan fluorescent proteins using nucleotide

sequences of genes. ------------------------------------------------------------------------------129

Fig. 7.6: Neighbor joining tree of Anthozoan fluorescent proteins using amino acid

sequences. -----------------------------------------------------------------------------------------130

Fig. 7.7: The predicted structure of HriGFP by using Phyre 2 in intensive mode.----- 131

Fig. 7.8: Location of fluorophore residues KYG in HriGFP structure, K is the part of α2,

while Y and G residues are located next to K residue between α2 and α3----------------132

Fig. 7.9: Comparison of protein structure of HriGFP and iLOV.---------------------------134

xxvi

Fig. 8.1: Soil sampling sites are indicated by red circles in the map of Pakistan

(Contaminations of these sites with methyl parathion are reported in previous studies--

139

Fig. 8.2: Pictorial view of some of sampled fields-------------------------------------------140

Fig. 8.3: Prevalence of various types of methyl parathion utilizing bacterial isolates (n= 64)

in soil samples of agricultural regions contaminated with methyl parathion. These isolates

grew in the presence of methyl parathion as sole source of carbon.------------- 149

Fig. 8.4: Prevalence (%) of heavy metal tolerance in methyl parathion utilizing bacterial

isolates (n = 64) -----------------------------------------------------------------------------------150

Fig 8.5: Antibiotic resistance pattern in methyl parathion utilizing bacterial isolates (n = 64)

---------------------------------------------------------------------------------------------------151

Fig 8.6: Integron and antibiotic resistance genes.----------------------------------------------152

Fig. 8.7: Prevalence (%) of antibiotic resistance genes (sul1, sul2, qnrA, qnrA, qnrB) and

integrons (int1, int2) in methyl parathion utilizing bacteria (n = 64) isolated in this study. -

-153

Fig 9.1: Cladogram (generated by MEGA 5 on the basis of 16S rDNA sequences) showing

relationship of bacterial species added in consortium. ---------------------------165

Fig. 9.2: Methyl parathion degrading activity of individual bacterial isolates and their

consortium.------------------------------------------------------------------------------------------166

Fig. 9.3: Growth curves of selected individual bacterial strains in MSM supplemented with

methyl parathion (0.6 mM methyl parathion). -------------------------------------------167

Fig. 9.4: Sol-gel with immobilized methyl parathion degrading Bacterial strains (X 8000

magnification) -------------------------------------------------------------------------------------168

Fig. 9.5: Stability of methyl parathion degrading consortium in suspension and TEOS based

sol-gel -----------------------------------------------------------------------------------------169

xxvii

Fig. 9.6: Stability of methyl parathion degrading consortium in suspension and sodium

silicate based sol-gel -----------------------------------------------------------------------------170

Fig. 9.7: Fluorescence microscopy of GFP expressing E. coli DH5α cells immobilized in

sol- gel matrices -----------------------------------------------------------------------------------171

Fig. 9.8: Light Microscopy of sol-gel immobilized GFP expressing bacterial cells (E. coli

DH5α) ----------------------------------------------------------------------------------------------172

LIST OF TABLES

Table 1.1: Toxic effects/ symptoms of methyl parathion and other organophosphate

pesticides (acute poisoning) in humans according to the sites of acetylcholine

accumulation --------------------------------------------------------------------------------------7

xxviii

Table 2.1: Primers used for amplification of PON1 coding region segments-----------17

Table 2.2: PCR conditions for amplification of PON1 coding region segments-------18

Table 2.3: Thermal cycling conditions for amplification of PON1 coding region

segments.-----------------------------------------------------------------------------------------18

Table 2.4: Allele and genotype frequencies of coding region polymorphisms of PON1

gene in Pakistani Punjabi healthy male population. ---------------------------------------24

Table 2.5: Combined genotypes frequencies of coding region polymorphisms of PON1

gene in Pakistani population-------------------------------------------------------------------25

Table 3.1: Demographic features of Pakistani Punjabi Pesticide workers--------------41


gene in healthy Pakistani Punjabi pesticide sprayers (n=100). ---------------------------50


gene in affected Pakistani Punjabi pesticide sprayers (n=101). -------------------------50

Table 3.4: Comparison of PON1 genotypes between controls and pesticide workers ---

-----------------------------------------------------------------------------------------------------51

Table 4.1: Blood chemistry and clinical data of the affected and normal individuals of

the studied family. ------------------------------------------------------------------------------68


gene in healthy controls of Punjabi hypercholesterolaemia family (n=24)--------------72


gene in hypercholesterolaemia patients of Punjabi family (n=10). ----------------------72

Table 4.4: Paraoxonase-1 allele and genotype distributions of L55M and Q192R

polymorphisms in patients and normal controls of hypercholesterolaemia family. ---73

Table 5.1: Clinical criteria of screening of study participants for glaucoma -----------91

xxix

Table 5.2: Demographic characteristics of study participants ----------------------------93


gene in age and gender matched healthy controls (non-glaucomic) (n=70).----------- 94


gene in POAG patients (n=90). ---------------------------------------------------------------95


gene in PCAG patients (n=60).--------------------------------------------------------------- 95

Table 5.6: Comparison of PON1 genotypes between controls and glaucoma patients.---

----------------------------------------------------------------------------------------------------97

Table 6.1: Organophosphate Binding sites (Y residues in vicinity of positively charged

amino acids) are shown as Y* within respective motifs of human and mouse

Transferrin--------------------------------------------------------------------------------------116

Table 7.1: Details of multiple templates used for prediction of 3D structure of HriGFP

by using Homology modeling method with the help of online tool (Phyre 2) in intensive

mode.-------------------------------------------------------------------------------- 123

Table 8.1: Levels of methyl parathion contamination reported at sampling location ----

---------------------------------------------------------------------------------------------------141

Table 8.2: Primers used for amplification of antibiotic resistance gene segments--------

---------------------------------------------------------------------------------------------------145

Table 8.3: Primers used for PCR-RFLP based identification of integrons (int1, int2,

int3)----------------------------------------------------------------------------------------------146

Table 8.4: PCR conditions for amplification of antibiotic resistance genes and integrons-

---------------------------------------------------------------------------------------147

Table 8.5: Thermal cycling conditions for amplification of antibiotic resistance genes

and integrons. ----------------------------------------------------------------------------------147

xxx

Table 9.1: Identification based on similarities of the 16S rDNA gene (partial sequence)

of methyl parathion degrading bacterial strains with previously known bacterial strains.-

------------------------------------------------------------------------------------------166

LIST OF ABBREVIATIONS

ACD Acid citrate dextrose (vacutainer)

AChE Acethylcholinesterase

AMP Ampicillin

xxxi

AS-III Arsenic (trivalent)

AS-V Arsenic (pentavalent)

Arg Argenine

ATPase Adenosine triphosphatase

BMI Body mass index

CAZ Ceftazidime

c/d Cup to disc ratio

CFU Colony forming units

ChE Cholinesterase

CIP Ciprofloxacin

CLSI Clinical and Laboratory Standards Institute

CN Gentamicin

CVD Cardiovascular disease

DLS Dynamic Light Scattering

DNA Deoxyribonucleic acid

DM Diabetes mellitus

dNTP Deoxyribonucleotide triphosphate

EDTA Ethylenediamine tetra acetate

FH Familial hypercholesterolemia

FPs Fluorescent Proteins

FT Fourier transform

xxxii

GC-MS Gas chromatography-mass spectroscopy

GFP Green Fluorescent Protein

Glu Glutamine

FPLC Fast Performance Liquid Chromatography

GIT Gastrointestinal tract

HDL High density lipoprotein

Hg Mercury

His Histidine

HPLC High performance liquid chromatography

HL Half life

HriCFP Cyan Fluorescent Protein of H. rigida

HriGFP Green Fluorescent Protein of H. rigida

IOP Intraocular presuure

IPTG Isopropyl Thio-Galactose

ITC Isothermal Titration Calorimetry

kDa kilo Dalton

LB Luria-Bertani

LD50 Lethal dose for 50% population

LDL Low density lipoprotein

LDLR Low density lipoprotein receptor

MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time of Flight

xxxiii

MDA Malondialdehyde

MIC Minimum inhibitory concentration

MN Micronucleus

MP Methyl Parathion

MS Mass spectroscopy

MSM Minimal Salt Medium

MTC Maximum tolerance concentration

NAL Nalidixic acid

NCBI National Center for Biotechnology Information

Ni-NTA Nickel-Nitrilotriacetic acid

NS Non significant

OAG Open angle glaucoma

OD Optical Density

OPIDP Organophosphate induced delayed polyneuropathy

OPs Organophosphate pesticides

ORF Open Reading Frame

PBS Phosphate buffered saline

PCAG Primary closed angle glaucoma

PCR Polymerase Chain Reaction

PDB Protein Data Bank

PMF Peptide mass fingerprint

xxxiv

POAG Primary open angle glaucoma

PON-1 Paraoxonase-1

PPEs Personal protective equipments

SDS-PAGE Sodium Dodecyl sulfate-polyacrylamide gel electrophoresis

sdLDL Small dense low density lipoprotein

SSR1 Selective serotonin reuptake inhibitor

SXT Sulphamethoxazole-trimethoprim combination

TA Annealing temperature

TC Total cholesterol

TG Triglycerides

Taq Thermus aquaticus

TE Tris-EDTA

TEOS Tetraethoxysilane

U Units

UV Ultraviolet

WHO World Health Organization

Y Tyrosine

CHAPTER 1: General Introduction and Literature Review

1.1 Methyl Parathion and Organophosphate Pesticides

1.1.1 Methyl parathion

xxxv

Methyl parathion (MP) is an organophosphate pesticide (OP) (Fig. 1.1). It has been

widely used due to its low cost and higher efficacy in pest control (Garcia et al., 2003). MP

is commonly used as an insecticide in production of cotton and other crops.

Methyl parathion and other OPs affect the target tissues by irreversibly acting as

AChE (acetylcholinesterase) inhibitor resulting in toxic effects due to accumulation of

acetylcholine (Vural, 2005). Methyl paraoxon inactivates AChE by phosphorilizing the

active region of AChE. This reaction remains reversible for a few hours only during the

initial chemical interaction. The reaction becomes irreversible after 24-48 hours due to

formation of strong covalent bonds which results in replacement of alkyl side chain of

AChE by a hydroxyl group. Re-activation of modified AChE becomes impossible after this

step (Dalkiliç, 2006).

Application of OPs in agriculture is associated with poisoning events (3 millions

per year) and death events (200,000 per year) in developing countries (Kwong, 2002).

Exposure to Folidol (active ingredient: methyl parathion) results in poisoning resulting in

deaths (Delen et al., 2005).

General Structure of OP Structure of Methyl Parathion

Fig. 1.1: General structure of OP compounds (R= methyl, ethyl or isopropyl, X = leaving

group) (Ref: Mohamed and Chia, 2008) (Molecular formula of methyl parathion:

C8H10NO5PS; Molecular weight: 263.21; commonly used names: parathion-methyl,

methyl parathion and metaphos) Common trade names: Metacid (previously Folidol)

1.1.2 Pharmacokinetics of methyl parathion

Humans and other living things are exposed to methyl parathion (and other OPs)

through different routes including contaminated air, soil, water and food (Fig. 1.2). Methyl

parathion is rapidly absorbed from skin, respiratory system and digestive tract in human

xxxvi

beings due to its lipid solubility. It is excreted mainly with urine (as p-nitrophenol)

(Yildirim, 2006). Tachycardia, dizziness, headache and heart failure were observed as

major effects in a death case associated with methyl parathion poisoning (WHO, 1993).

Methyl parathion is rapidly distributed in living organisms after absorption. Methyl

parathion is detected in blood plasma of rats after 6-8 minutes after oral administration.

MP was detected in brain tissue of rats after 90 seconds following intravenous

administration (Yamamoto et al., 1983). Ninety percent of plasma AChE activity in the

fish (Callichthys callichthys) is inhibited after 4 h following the administration of Folidol

600 (active ingredient: methyl parathion) and this inhibitory effect continued to persist up

to 4 days (Da Silva et al., 1993).

Methyl parathion is rapidly absorbed in chicken by GIT (gastrointestinal tract) and

spreads to various organs (liver, kidney, brain). Methyl parathion was detected in liver of

chicken after eight hours following its oral administration (Abu-Qare and Abou-Donia,

2001). All of these pharmacokinetic properties of methyl parathion ultimately contribute

to un-intentional poisoning events in the non-targeted organisms (organisms other than

targeted pests).

1.1.3 Methyl parathion toxicity revealed by animal models

Most of the data about toxic effects of methyl parathion is generated by animal

studies which revealed a very wide range of toxic effects of methyl parathion. Decrease in

body weight of experimental animals associated with toxic effects of methyl parathion is

reported in some studies (Kahraman 2006). Body weight of rats is reported to be decreased

after one week of dermal administration of methyl parathion (1 mg/kg) (Zhu et al. (2001).

A significant decrease in sulphydryl group due to oxidative stress in liver and kidney

proteins in rats exposed to methyl parathion for three months was reported by Dalkilic

(2006).

xxxvii

Fig. 1.2: Different routes of exposure to methyl parathion and other Organophosphate

pesticides (Adapted from WHO, 2001)

xxxviii

Increased serum LDH levels in the rats exposed to methyl parathion was reported

by Dikshith et al., (1991). An increased liver weight associated with Dimethoate (methyl

parathion) and dose dependant reduction in erythrocyte cellular volume was reported by

Undeger et al. (2000) in Wistar rats after 28 days oral administration of Dimethoate.

Reproductive system is also adversely affected by methyl parathion. Reduction in

ovarian weight as a toxic effect of methyl parathion was reported by Asmathbanu et al.,

(1997). Induction of estrogen cycle disorders due to methyl parathion was reported by

Sortur and Kaliwal, 1999. A dose dependant increase in number of abnormal sperm cells

was reported in rats as a toxic effect of methyl parathion (Mathew et al., 1992). Toxic

effects of methyl parathion in male reproductive system, including decreased weight and

degeneration of testicles, were reported in rats by Joshi et al. (2003). Bartoli et al., (1991)

described DNA damage in rats as a toxic effect related to methyl parathion. Methyl

parathion induced increased chromosome abnormalities were reported by Bartoli et al.,

1991. A cytotoxic effect of methyl parathion on male reproductive cells in rats was studied

in detail by Narayana et al., (2005). They detected decrease in total sperm content and

increased abnormal sperm count as toxic effects of methyl parathion. It is also interesting

to note that male rats were allowed to match with the control group female rats and a

decrease in body weight of newborn babies was also detected in this experiment (Narayana

et al., 2005). A decrease in mobility as well as total number of sperms with increased

number of morphologically defected sperms due to methyl parathion toxicity was observed

in a study by Uzunhisarcikli et al. (2008). Dermal application of methyl parathion resulted

in inhibition of AChE activity in pregnant rats as well as their fetus (Abu-Qare and Abou

Donia 2001).

Different biochemical processes are also widely affected by methyl parathion. In

one of the animal models, Guney et al., (2007) reported an increase in malondialdehyde

(MDA) and decreased cholinesterase (ChE) activity associated with methyl parathion

administration. The opposite effects of decrease in MDA and increase in ChE activity was

detected with administration of methyl parathion along with combination of vitamin E and

C. A decrease in blood ChE activity was also detected in rats by Ho et al. (2001) due to

methyl parathion administration. Inhibition of oxidative phosphorylation due to methyl

xxxix

parathion and decreased total adenosine triphosphatase (ATPase) at tissue level was

reported by Rao and Rao (1984). Decreased weight of adrenal gland associated with methyl

parathion toxicity was reported in rats (Liu et al., 2006). Cypermethrin and methyl

parathion combination was administered to rats in this study. Pathological changes

including necrosis and glomerular atrophy due to methyl parathion in male rats was

observed by Yildirim (2006) in kidney tissues. All of these reports suggest a very wide

range of toxic effects of methyl parathion in a living body.

1.1.4 Effects of environmental contamination with methyl parathion

Although some studies have revealed important information about effects of methyl

parathion on ecosystem, however the complete picture is still not well understood. Methyl

parathion persists in the environment for up to a few weeks or a few months after its

application in agricultural fields (ATSDR, 1990).

Presence of methyl parathion in applied region contaminates human beings and

other species. Non-target organisms, vertebrates as well as invertebrates, are adversely

affected with toxic effects of methyl parathion due to environmental contamination (Fanta

et al., 2003). LD50 values of methyl parathion reported for living organisms, for example

bees (0.17 mg), birds (3 and 8 mg/kg body weight) and fish (6 - 25 mg/L) are very low and

hence it is considered an important environmental contaminant (IPCS, 1993). Methyl

parathion is also very toxic to mammals. Oral, dermal and respiratory routes are the main

routes of exposure to methyl parathion in the mammalians (Garcia et al., 2003).

Aquatic life can suffer a great damage due to contamination of water with methyl

parathion. Histopathological changes in liver, gills and intestine of Corydoras paleatus fish

due to their exposure to low (sub-lethal) concentration of methyl parathion in contaminated

food and water were reported by Fanta et al. (2003). In one of the studies, De Aguiar et al.,

(2004) reported 64% reduction in AChE activity in plasma and 87% reduction in brain of

the Brycon cephalus fish which were exposed to Folidol 600 (methyl parathion). A

decrease in ALT (59.4% reduction) in liver and 92.2% increase in ALT in plasma was also

detected. Tissue damage in heart was also indicated in fish in this study by an increased

AST activity.

xl

Bianchini and Monserrat (2007) studied effects of methyl parathion on a crab specie

called yengeclere (Chasmagnathus granulatus). It is the only specie in the genus Neohelice

of the family Verunidae. The crabs of this unique specie were exposed to methyl parathion

(10/100 LD50) for 72 hours in an experiment. Hepatopancreatic tubules of crabs were

adversely affected due to methyl parathion. Level of antioxidant enzymes activity was

enhanced alongwith increased level of lipid peroxidation in hepatopancrease of the crabs

injected with methyl parathion.

Birds are especially prone to adverse effects of methyl parathion in case of

environmental contamination. Effects of methyl parathion were investigated in Lonchura

malabarica (a small bird called Indian Silverbill, found in the regions of Indian

subcontinent) in a study by Maitra and Sarkar (1996). The birds were exposed to sub-lethal

dose of methyl parathion (5 to 20 μg/100 g body weight) by oral administration. A dose

dependant decrease in AChE activity of brain tissues and testicles was detected. In addition

seminiferous tubules were affected in terms of decrease in normal reproductive cells count

after 10 days after methyl parathion administration. A decrease in the ChE level of brain

and plasma was observed in quails when they were administered with 14 to 40 ppm of

methyl parathion for a period of six weeks. In addition Weight of their eggs and the egg

shells’ thickness was reduced (Maitra and Sarkar, 1996). All of these previous studies

indicate highly toxic effects on the living organisms due to environmental contamination

with MP.

1.1.5 Methyl parathion toxicity in humans

Methyl parathion is one of the highly toxic chemicals for humans. MP can not be

washed away from the human skin after 15 min of exposure (Vural, 2005). Absorption of

parathion from skin during application can result in poisoning effects which may be acute

or even lethal (Vural, 2005). Blasiak and Kowalik, (1999) reported genotoxic effects of

methyl parathion in human lymphocytes. Changes in EEG (electroencephalogram) pattern

of farmers exposed to methyl parathion (due to contaminated water or spray) indicated

AChE inhibition in brain in one of the studies (Muttray et al., 2005). Cholinesterase activity

of erythrocytes was decreased by 15% in farmers who applied methyl parathion by spray

for 2 hours (He et al., 2002). Methyl parathion was reported by Barr et al., (2005) as one

xli

of the most common pesticides to which general population was exposed which indicates

that general population is also at an increased risk of toxicity due to methyl parathion.

Usage of methyl parathion in household cleaning also leads to affect the exposed

individuals. As a result, people exposed to methyl parathion suffer from various levels and

types of ill health effects due to toxicity of methyl parathion (Leveridge, 1998). Sickness

in children and depression in adult humans is documented as adverse effects of long term

methyl parathion exposure in America at domestic and farm level (Rehner et al., 2000).

Some of the major signs and symptoms of toxic effects of methyl parathion previously

reported in humans are listed in Table 1.1.

Table 1.1: Toxic effects/ symptoms of methyl parathion and other organophosphate

pesticides (acute poisoning) in humans according to the sites of acetylcholine accumulation

Type of OP Toxicity

Effects (signs and symptoms)

Muscarinic actions

Nicotinic actions

Central nervous system

Other effects

Sweating, tear and stenosis in bronchus, excessive

salivary secretion, nausea, vomiting, diarrhea,

abdominal pains as cramps, urinary and stools

incontinence, bradycardia

Uncontrolled muscle contractions, twitches,

paralysis

Headache, insomnia, vertigo, spasms, confusion,

speaking disorders, excess discontent,

nervousness, dizziness, dullness, decline in

memory, depression, sudden and uncontrolled

movements, coma and respiratory standstill.

xlii

Destruction of membrane structures of lysosomes

(in lenfosides), immunotoxicity (by inhibiting the

lenfoxin secretion)

References: (Sharma and Reddy, 1987; Tafuri and Roberts, 1987; Taylor, 1996;

Lefkowitz et al., 1996, Tuovinen, 2004)

1.1.6 Current analytical techniques for methyl parathion detection

Although current techniques of methyl parathion detection (Gas chromatography,

liquid chromatography, etc.) are very sensitive as well as reliable, however they are not

suitable for field studies. These techniques require skilled manpower, lengthy analytical

procedures and high cost of analysis which make these techniques inaccessible for most of

the remote agricultural areas in the developing countries with poor economy. Thus portable

biosensor systems are being developed for the monitoring of OPs in the real field

conditions in agricultural areas and other environmental segments.

Some biological methods for detection of OPs have been developed on the basis of

immunoassays and cholinesterase inhibition activity (Sherma, 1993; Trojanowicz and

Hitchman, 1996). Biosensors based on AChE inhibition test are sensitive but they have

limitations of inhibition of AChE by several other chemical agents. Potentiometer

transducers have also been developed which detect the substrate on the basis of measuring

xliii

the pH changes due to acetic acid production during test procedure in the presence of target

OP substrate (Dzyadevich et al., 1994). Fiber optic technology is also being tested for

biosensor applications which measure the pH changes during sample analysis (Moris et al.,

1995).

OP biosensors are also developed on the basis of organophosphorus hydrolase

activity of enzymes of bacterial origin. These enzymes hydrolyze a wide range of OPs

including parathion (Dumas et al., 1990). Electrons liberated during hydrolysis of OPs due

to breaking of chemical bonds are electroactive which can be detected as the signal for

presence of substrate (OP) in the test samples. Strength of electrical signals produced

during hydrolysis can be used as a measure of concentration of the OP substrates (methyl

parathion and others). These biosensor studies have attempted to minimize the need of

sophisticated equipments. However, still further studies are needed to produce simpler and

portable biosensors for detection of OP pesticides in the real field conditions in remote

areas.

1.2 Paraoxonase 1 (PON1)

The human paraoxonase 1 (PON1) is an enzyme (a calcium-dependent esterase)

which has multiple functions including hydrolyses of toxic metabolites of methyl parathion

and other OPs. PON1 gene is located at long arm of chromosome 7 (q21.22) in case of

humans (Fig. 1.3). PON1 protein structure consists of six-bladed β-propeller with a central

tunnel of the propeller containing calcium atoms (Ca1 and Ca2) (Fig 1.4). This protein is

composed of 354 amino acids (45 kDa) (Primo-Parmo et al., 1996). It is synthesized in the

liver. After secretion into the blood stream, it becomes associated with serum HDL

(Mackness et al., 1998).

1.2.1 PON1 gene polymorphisms

Number of polymorphisms of PON1 gene identified till now is about 200. These

polymorphisms occur in some of the coding regions, introns and promoter regions (Jarvik

et al., 2003). Wide interindividual and interethnic variations in PON1 activity have been

related to PON1 SNPs. Two most significant and most widely studied SNPs are located in

xliv

the PON1 coding region: Q192R polymorphism at position 192 and L55M polymorphism

at position 55 (Costa et al., 2005).

1.2.1.1 Q/R polymorphism in coding region (position 192)

Efficiency of PON1 as an enzyme is significantly affected by PON1 Q192R

polymorphism. In vitro studies have demonstrated that enzymatic activity of two isoforms

of PON1 with respect to position 192 (Glu/Arg) is substrate dependant. The 192R allozyme

is more efficient in hydrolyzing paraoxon (Costa et al., 2005) while 192Q allozyme

hydrolyze diazoxon and related compounds (sarin and soman) more rapidly (Davies et al.,

1996). Phenylacetate is hydrolyzed by both isoforms (192Q and 192R) at similar rates

under physiological conditions (La Du et al., 1986, Li et al., 2000).

1.2.1.2 PON1 L/M polymorphism

Catalytic activity of PON1 is not affected by the polymorphism at position 55

(L55M). However plasma PON1 levels are largely determined by PON1 L/M

polymorphism. The M55 is associated with low levels of plasma PON1 as compared to

L55 (Mackness et al., 1998). Studies suggest that this association of L55M with PON1

levels is due to linkage disequilibrium with the promoter region polymorphism (Brophy et

al., 2001), In addition, the 55L isoenzymes is more stable than 55M (Leviev and James,

2000).

xlv

Fig. 1.3: PON1 gene structure. (A): PON1 gene family located on chromosome 7. (B):

PON1 gene and its commonly studied polymorphisms (Adapted from Furlong et al., 2002)

Fig 1.4: Structure of PON1 protein. (a): View of crystalline structure of PON1 from above.

PON1 is made up of six-bladed β-propeller (A-D). The N and C termini and the two

calcium atoms in the central tunnel of the propeller (Ca1 green, Ca2 red) are shown. (b): A

side view of the propeller including the three helices at the top of the propeller (H1-H3).

(Adapted from Harel et al., 2007).

1.2.2 Physiological roles of PON1 protein

xlvi

Natural substrate of PON1 is still not known. However it hydrolyzes toxic

metabolites of methyl parathion and other OPs (Billecke et al., 2000). Furthermore, the

enzymatic efficiency of PON1 protein for hydrolyzing these toxic compounds varies

depending on the particular substrate (Costa et al., 2005). Variations in serum PON1

activities are associated with genetic makeup and some environmental factors (Senti et al.,

2003).

PON1 plays a significant role in lipid metabolism, especially in hydrolyzing lipid

peroxides, preventing LDL oxidation, inactivating oxidized phospholipids and preventing

oxidation of phospholipids (Costa et al., 2003). These physiological roles of PON1 help in

prevention of atherosclerosis development (Durrington et al., 2001). Human paraoxonase

(PON1) is associated to HDL particles (Brophy et al., 2000). A relationship between PON1

polymorphism and an enhanced risk for coronary artery disease has been detected in

previous studies (Draganov and La Du, 2004). In some of the studies it has been found that

PON1 acts as a protector agent against the development of the atherogenic plaque due to

its capacity of hydrolyzing oxidized lipids present in the arterial wall (Durrington et al.,

2001). It is well known that the accumulation of oxidized LDL particles is one of the factors

involved in heart diseases as a consequence of development of atherosclerosis. PON1

stimulates HDL mediated macrophage cholesterol efflux and protects LDL from oxidation

(Rosenblat et al, 2006). Thus collectively, PON1 plays a protective role against

development of atherosclerosis.

In addition to prevention of atherosclerosis PON1 has also been found to be

associated with other biological processes in human body. For example, oxidative stress is

reduced by PON1 which ultimately regulates aging process (Marchegiani et al., 2008). On

the other side, PON1 levels and its activity are increased in pregnancy and during delivery

(Huen et al., 2010). PON1 level is low in infants due to which they have low antioxidant

capacities (Huen et al., 2010). PON1 is also known to be associated with diabetes mellitus

(DM) (Hofer et al, 2006). An association between insulin resistance and reduced PON1

activity has been reported (Hedrick et al., 2000). Decreased PON1 activity has been

detected in patients with diabetes compared to healthy controls (Mastorikou et al, 2006).

Contribution of PON1 has also been investigated in many other disease conditions

including stroke, coronary artery disease, hypertension, Parkinson’s’ disease, etc. Thus it

xlvii

is apparent that PON1 plays important role(s) in multiple of processes in health and disease

by performing various functions in different organs including brain, lungs, liver and

intestine, etc. (Fig 1.5).

Brain: Link between PON1

polymorphism and Parkinson’s

disease

Blood circulation: PON1 protein is

transported by HDL in circulation

and prevents LDL and HDL from

oxidation in endothelial cells

Lungs: Expression of PON1 protein is

important for the control of bacterial

quorum sensing

Liver: synthesis of PON1 protein occurs in

liver. PON1 enhances liver’s capacity for

anti-oxidative and anti-inflammatory

defense

Intestine: Expression of PON1 produces

anti-oxidative and anti-inflammatory

effects

xlviii

Fig. 1.5: Some important physiological effects of PON1 protein on major organs (Adapted

and modified from Precourt et al., 2011)

1.3 Problem statement

As methyl parathion is a highly toxic substance widely used in pest control so it is

necessary to identify the human populations susceptible to toxic effects of methyl

parathion. In addition, detection of methyl parathion in the environment can be helpful for

safeguarding humans and other non-targeted living organisms. In field and large scale

detection of methyl parathion would become more feasible and less costly if new and

simplified methods of methyl parathion are developed. Finally, decontamination of the

contaminated environmental and agricultural segments would become more “environment

friendly” and less damaging to the natural system if natural resources capable of degrading

methyl parathion are explored and studied.

1.4 Objectives of the study

Following are the main objectives which we have focused in our studies reported

in this thesis:

1- To determine the normal baseline values of serum paraoxonase and arylesterase

activities and related genotypes in healthy Pakistani individuals of the largest ethnic group

of Pakistan exposed to pesticides

2- To determine pesticide susceptibility patterns and underlying factors predisposing

pesticide workers to adverse effects of pesticides

3- To study any other significant associations of PON1 phenotype and genotype with

related diseases in Pakistan (Glaucoma and Hypercholesterolaemia)

4- To explore simple and economical method for detection of methyl parathion by using

fluorescent protein

5- Characterizing novel fluorescent protein (HriGFP) isolated from Hydnophora rigida

(Horn coral)

6- To identify best possible naturally occurring bacterial isolates capable of utilizing

methyl parathion as a source of nutrient

xlix

7- To explore utility of sol-gel matrices for supporting long term survival of methyl

parathion utilizing bacterial isolates

(Case Study – 1)

2.0 Paraoxonase-1 Gene Polymorphism in Punjabi Population of

Pakistan

2.1. Introduction

Human paraoxonase 1 (PON1) enzyme is a multitasking protein. The PON1 protein

has been reported to be involved in a multiple of functions in health and disease including

the genetic susceptibility to toxicity with methyl parathion and other organophosphate

pesticides (Davies et al., 1996). In addition, it plays a vital role in different diseases

(Marchegiani et al., 2008), atherosclerosis (Durrington et al., 2001), protection against

carcinogenic effects of ionizing radiation (Serhatlioglu et al., 2003), protection against

aging process and oxidative stress (Marchegiani et al., 2008), protection against

acetaminophen intoxication (Karadas et al., 2014), variability of response to therapeutic

agents (Xie et al., 2014) and susceptibility to toxicity of warfare agents (cyclosarin and

other nerve agents) (Worek et al., 2014), etc. Due to involvement of PON1 in multiple of

health and disease processes, it can be expected that the data generated by population based

study of PON1 gene polymorphism and activity would be very significant for public health

improvement at gross levels.

Similarities of PON1 polymorphism between related ethnic groups have been

documented in some of the studies (Malin et al., 2001). On the other hand, ethnic

differences in PON1 polymorphism (Poh and Muniandy, 2007) and PON1 enzyme

activities (Davis et al., 2009) have also been highlighted previously. For example, meta-

analysis of case control studies (You et al., 2013) indicated that PON1 192Q and 192L

polymorphisms are associated with increased susceptibility to OP toxicity. This association

was significant in Caucasian populations while the same association was not clear in Asian

l

populations (You et al., 2013). Thus role of PON1 needs to be explored further on the basis

of ethnicity in order to explain any similarities or differences between different

populations, especially in Asian populations where the role of PON1 has not been fully

explored and the previous studies in Asian populations are inconclusive (You et al., 2013).

There are only two reports (Saeed et al., 2007 and Batool et al., 2014) about PON1

in Pakistan to the best of our knowledge. Saeed et al., (2007) reported association of PON1

polymorphism with myocardial infarction, whereas Batool et al., (2014) studied

association of diabetes with PON1 and other genes. Both of these studies lack any

information about PON1 enzymatic activities without which the role of PON1 can not be

studied in conclusive way because previous studies which covered only genotypes (without

PON1 protein activities) revealed inconclusive and contradictory data. Considering that

little or nothing is known about PON1 genotypes and enzyme activity in general population

of Pakistan, we investigated the frequency of the PON1 Q192R and PON1 L55M

polymorphisms alongwith enzyme activity (serum paraoxonase and arylesterase) in a

Pakistani Punjabi population. Results were compared with data reported in other studies to

find out the variation from other populations. The results will be valuable in finding out

physiological range of PON1 activity and risk of various diseases in adult healthy Punjabi

population of Pakistan.

li

2.2. Materials and Methods

2.2.1 The study population

Randomly selected healthy Punjabi volunteers (n = 500) were interviewed for

inclusion of participants based on their current and past health status, age and lifestyle

habits. Exclusion criteria included alcohol and tobacco consumption and presence of any

disease within a period of previous six months. Blood samples were collected from 140

short listed Punjabi male healthy participants with 23−40 years of age. All of these sampled

persons were unrelated healthy volunteers (non obese, non smokers, non alcoholic, not on

any medication, no hypertension, non diabetic, non hypercholesterolemic, non

hyperlipidemic and no history of exposure to pesticides). The study was approved by the

Ethics Committee of the Department of Biosciences, CIIT, Islamabad. Written informed

consent was obtained from participants.

2.2.2 Samples

Blood samples (5 mL) were collected in separate tubes for serum separation (for

serum paraoxonase and arylesterase activities) and DNA extraction (Helms, 1990). For

DNA extraction, blood was collected in Acid Citrate Dextrose (ACD) vacutainer (Becton–

Dickinson product no. 364606, Franklin Lakes, NJ) and for serum separation blood was

collected in Z serum sep Clot Activator vacutainer tubes (Greiner bio-one, Munich,

Germany). Serum was separated from clotted blood by centrifuging the vacutainers at 3000

rpm for 10 min at 4 oC.

lii

2.2.3 Determination of PON1 SNPs

Genomic DNA was extracted from peripheral blood leukocytes using standard

procedures (Helms et al., 1990). Two sets of primers were used for genotyping the

polymorphisms of codon 192 and codon 55 in the PON1 gene as described by Motti et al.,

2001. The reverse primers contained mismatched nucleotides (as indicated by lower case

letters in italics in primer sequences. Table 2.1). This allowed a restriction site for HinfI

(G/ANTC) to be introduced into the DNA amplification products in the presence of the

polymorphisms arginine-PON1-192 or leucine-PON1-55. Reaction conditions for PCR

amplification of PON1 coding region segments are described in Table 2.2. and Table 2.3.

PCR products were purified using the DNA extraction kit (Fermentas) and subjected to

restriction with Hinf1 enzyme (Fermentas) to screen for the type of SNP in the target

sequence. The results were analyzed to determine the prevalence of PON1 L55M and

PON1 Q192R SNPs.

Table 2.1: Primers used for amplification of PON1 coding region segments

Primer

name

Primer sequence (5’-3’) TA Product

size

Hinf1 treated

PCR product

PON1

Q192R-

forward

TTGAATGATATTGTTGCT

GTGGGACCTGAG

57 oC 111 bp

QQ: 111 bps

QR: 111 bps,

77 bps

RR: 77 bps

PON1

Q192R-

reverse

CGACCACGCTAAACCCAA

ATACATCTCCCAGagA

(due to this mismatch primer,

nucleotide G of the reverse

strand of gene is replaced with

A in PCR product creating a

liii

restriction site for Hinf1 in case

of R-192 allele)

PON1

L55M-

forward

GAGTGATGTATAGCCCCA

GTT TC

57 oC 144 bp

MM: 144 bps

LM: 144 bps,

122 bps

LL: 122 bps

PON1

L55M-

reverse

AGTCCATTAGGCAGTATC

TCCga (due to this mismatch

primer, nucleotide A of the

reverse strand is replaced with

G creating restriction site for

Hinf1 in PCR product in case of

L-55 allele)

TA: Annealing temperature used for PCR amplification

Table 2.2: PCR conditions for amplification of PON1 coding region segments

Components Concentration

dNTPs

PCR buffer

MgCl2

Each primer (forward/reverse)

Taq. Polymerase

Genomic DNA

0.3 mM

1x (10 mM Tris–HCl pH 9.0, 50 mM KCl)

2.0 mM

0.5 mM

1.5 U

50 ng

Table 2.3: Thermal cycling conditions for amplification of PON1 coding region segments.

liv

Step Temperature Time

1- Initial denaturation

2- Denaturing

3- Primer annealing

4- Chain extension

5- Final extension

95 oC

95 oC

57 oC

72 oC (Go to step 2; 35 × )

72 oC

4 min

45 sec

1 min

1 min

7 min

2.2.4. PON1 paraoxonase activity measurement

The paraoxonase activity was determined by measuring the absorbance at 412 nm

(using paraoxon as substrate) due to the formation of 4-nitrophenol (Zehra et al., 2009).

The assay mixture contained 1.0 mM paraoxon, 1.0 mM CaCl2, 50 mM glycine/NaOH

buffer (pH 10.5). The amount of liberated 4-nitrophenol was calculated on the basis of its

molar coefficient 18,290/Mcm. Results of serum paraoxonase activities were recorded as

U/L. All samples were tested in triplicate.

2.2.5. PON1 arylesterase activity measurement

Arylesterase activity was determined as an indicator of PON1 protein concentration

in serum (Garin et al., 1997) by using an assay mixture containing 1.0 mM phenylacetate,

0.9 mM CaCl2, 20 mM Tris-HCl buffer (pH 8.0). Rate of hydrolysis was determined at 270

nm. The results were calculated by using extinction coefficient 1310/Mcm. Results of

serum arylesterase activities were recorded as U/mL. All samples were tested in triplicate.

2.2.6. Statistical analysis

Allele and genotype frequencies were calculated by gene counting. Results for all

continuous variables are expressed as Mean ± SD. Differences among different groups

(cases vs controls) were assessed by using Student’s t-test, χ2 test and Fisher’s Exact test.

lv

(The Student t-test was used to compare group means of the studied parameters.

Categorical variables were compared by the χ2 test and Fisher’s Exact test). Statistical

analysis was done by using SPSS (version 17) Differences between genotype distributions

of patients and controls were evaluated using the chi-square test. Allele distribution

differences of controls and patients were evaluated by the Fisher exact test. P values less

than 0.05 were considered as statistically significant.

2.3. Results

2.3.1. Demographic properties

PON1 genotype and serum paraoxonase and arylesterase activities of 140 non-

related volunteers were determined. All the participants were male (n = 140), with Mean

age of 30±10 years and Mean BMI 24.50±3.26 kg/m2.All the participants were in their

sound health with no apparent health problem on clinical examination. All of them were

normotensive for their age and body weight. None of them was on any type of medication

since previous six months. All the participants were non-alcoholic and non smokers.

lvi

2.3.2. PCR-RFLP

Respective band sizes of PCR and RFLP products (Fig. 2.1- 2.4) were in

accordance with previously reported (Motti et al., 2001) respective band sizes. PCR for

amplification of PON1 Q192R resulted in 111 bps bands and PON1 L55M was amplified

as 144 bps segment. Restriction of PON1 Q192R PCR product resulted in 111 bps for QQ,

111 and 70 bps segments for QR and 70 bps for RR (Fig. 2.2). Restriction of PON1 L55M

PCR product resulted in 144 bps for MM, 122 and 22 bps for LM and 122 bps segments

for LL genotypes (Fig. 2.4).

Fig. 2.1: PCR amplified products of PON1 Q192R (111 bps). Lanes 1-10: PCR product,

M: DNA marker

1 2 3 4 5 6 7 8 9 10 M

111 bps

(bps)

1500

1000

500

400

300

200

100

lvii

Fig. 2.2: PCR-RFLP results of PON1 Q192R. (QQ is represented by 111 bps single band;

QR showed 111 bps and 70 bps bands; RR was identified on the basis of single band of 70

bps). Lanes 1-8: Hinf1 restricted PON1 Q192R PCR products. M: DNA marker

M 1 2 3 4 5 6 7 8 M bps

500

400

300

200

100

bps

500

400

300

200

100

111 bps

70 bps

lviii

Fig. 2.3: PCR amplified product of PON1 L55M (144 bps). M: DNA marker, Lanes 1-5:

PCR products (144 bps)

1 2 3 4 5 M

PON1

L55M

(144 bps)

bps

1500

1000

500

400

300

200

100

lix

MM LM LL LL LL LL LL LL LL LL LL LL LL LM LM

Fig. 2.4: PCR-RFLP results of PON1 L55M. (Respective band sizes for genotypes are as

follows: MM- 144 bps single band; LM-144 bps and 122 bps bands; LL-122 bps single

band). Lanes 1-15: Hinf1 restricted PON1 L55M PCR products; M: DNA marker

2.3.3. Allele and genotype frequencies

144 bps

122 bps

bps

1500

500

400

300

200

144

122

100

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

lx

Allele and genotype frequencies for PON1 Q192R and PON1 L55M were

determined on the bases of PCR-RFLP patterns. The results of frequencies are summarized

in Table 2.4. Our results indicate that Q allele (69%) is predominant over R allele (31%)

and L allele (74%) is predominant over M allele (26%) in the studied healthy Punjabi

population. Genotype QQ (54%) and LL (53%) were predominant over remaining

genotypes (QR, RR, LM and MM).

Table 2.4: Allele and genotype frequencies of coding region polymorphisms of PON1 gene

in Pakistani Punjabi healthy male population.

Polymorphism Allele

n = 280

Frequency Genotype

n = 140

Frequency

n % n %

PON1 Q192R Q

R

193

87

69

31

QQ

QR

RR

76

43

21

54

31

15

PON1 L55M L

M

207

73

74

26

LL

LM

MM

74

59

7

53

42

5

2.3.4 Frequencies of combined genotypes

Frequencies of combined genotypes of PON1 Q192R and PON1 L55M in healthy

male Punjabis are summarized in Table 2.5 and further elaborated in Fig. 2.5. The results

indicate that QQ/LL (24.29%), QQ/LM (25%) and QR/LL (19.28%) were predominant in

studied population as compared to other combined genotypes. The prevalence of RR/MM

(1.43%), QR/MM (1.43%) and QQ/MM (3.57%) was low in the studied population as

compared to other combined genotypes.

lxi

Table 2.5: Combined genotypes frequencies of coding region polymorphisms of PON1

gene in Pakistani population

Combined genotype Frequency

n %

QQ/LL

QQ/LM

QQ/MM

QR/LL

QR/LM

QR/MM

RR/LL

RR/LM

RR/MM

34

35

5

27

14

2

12

9

2

24.29

25

3.57

19.28

10

1.43

8.57

6.43

1.43

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

lxii

Fig. 2.5: A 3D chart showing pattern of relative frequencies (%) of combined genotypes

of PON1 L55M and PON1 Q192R genotypes in healthy male Punjabis (n = 140) with no

history of exposure to pesticides.

2.3.5. Serum paraoxonase activities according to PON1 L55M and PON1 Q192R

Serum paraoxonase and arylesterase activities in healthy Punjabis were determined

and arranged according to genotypes of the individuals. Paraoxonase activities were

significantly different between individuals with various combinations of PON1 L55M and

PON1 Q192R alleles. RR genotype was associated with highest (460±98 U/L) and MM

genotype was associated with lowest serum paraoxonase activity (94±10 U/L). Serum

paraoxonase activity was higher in RR genotype (460±98 U/L) than QR (297±53 U/L) and

QQ genotype individuals (150±20 U/L) (RR>QR>QQ). On the other side serum

paraoxonase activity was higher in LL genotype (284±42 U/L) as compared to LM (198±21

U/L) and MM genotype (94±10 U/L) individuals (LL>LM>MM). Serum paraoxonase

activities showed significant differences between individuals of different genotypes of

PON1 L55M and PON1 Q192R (P<0.05) (Fig 2.6).

0

100

200

300

400

500

600

700

QQ QR RR MM ML LL

Genotype

Sp

ec

ific

ac

tiv

ity

(M

ea

n)

a

a

a

b

b

b

Serum

paraoxonase

activity

(U/L)

lxiii

Fig. 2.6: Serum paraoxonase activity (U/L) of Pakistani Punjabi healthy male individuals

(with no exposure to pesticides, n = 140) with respect to PON1 L55M and PON1 Q192R

genotypes. Results are shown as Mean ± SD. a: indicates statistically significant

differences (P<0.05) of serum paraoxonase activities between PON1 Q192R genotypes. b:

indicates statistically significant differences (P<0.05) of serum paraoxonase activities

between PON1 L55M genotypes


Serum arylesterase activity was less variable (62±13 U/mL to 136±29 U/mL) as

compared to paraoxonase activity of the individuals with respect to PON1 genotypes.

Differences of arylesterase activities between QR, RR and QQ individuals were not

significant. However significant differences (P<0.05) were found between the serum

arylesterase activities of individuals with LL, LM and MM genotypes. Serum arylesterase

activity increased in the following order with respect of each genotype: QQ>QR>RR and

MM>ML>LL (Fig. 2.7).

0

20

40

60

80

100

120

140

160

180

QQ QR RR MM ML LL

Genotype

Sp

ec

ific

ac

tiv

ity

(M

ea

n)

NS

b

b

b NS

NS

Serum

arylesterase

activity

(U/mL)

lxiv

Fig. 2.7: Serum arylesterase activity (U/mL) of Pakistani Punjabi healthy male individuals

(with no history of exposure to pesticides) (n = 140) with respect to PON1 L55M and PON1

Q192R genotypes. Results are shown as Mean ± SD. NS: statistically non significant

differences of serum arylesterase activities between PON1 Q192R genotypes. b:

statistically significant differences (P<0.05) of serum arylesterase activities between PON1

L55M genotypes

2.3.7. Combined results of paraoxonase and arylesterase activities according to

PON1 L55M and PON1 Q192R

Combining the results of paraoxonase and arylesterase activities together according

to respective genotypes (Fig. 2.8) revealed that serum paraoxonase activity was highly

variable compared to serum arylesterase activity of individuals with different genotypes in

healthy male Punjabi population (Fig. 2.8).

0

100

200

300

400

500

600

700

QQ QR RR MM ML LL

Genotype

Sp

ec

ific

ac

tiv

ity

(M

ea

n)

Paraoxonase U/L

Arylesterase U/ml

Serum

paraoxonase

and

arylesterase

activities

lxv

Fig. 2.8: Serum paraoxonase and arylesterase activities of healthy male Punjabis according

to presence of different genotypes (PON1 Q192R polymorphism: QQ, QR, RR and PON1

L55M polymorphism: LL, LM, MM) of coding region polymorphisms of PON1 gene.

2.3.8. PON1 Enzyme activities with respect of combined genotypes

Results of serum paraoxonase and arylesterase activities were further arranged

according to combined genotypes of PON1 Q192R and PON1 L55M (Fig 2.9). Highest

serum paraoxonase activity was observed in RR/LL (427±97 U/L) and lowest in QQ/MM

genotype (96±10 U/L). Highest serum arylesterase activity was observed in QQ/LL

(148±20 U/mL) and lowest in RR/MM genotypes (56±8 U/mL).

lxvi

Fig. 2.9: Serum paraoxonase (U/L) and arylesterase (U/mL) activities in healthy male

Punjabis with no history of exposure to pesticides (n = 140) according to combined

genotypes of coding region polymorphisms of PON1 gene.

2.4. Discussion

This is one of the first report on PON1 coding region polymorphisms along with

enzyme activity in healthy male Pakistani Punjabi population which has no history of

exposure to pesticides. The significant differences between serum paraoxonase activities

of various genotypes indicated that paraoxonase activity is associated with PON1 coding

region SNPs in healthy population of single ethnic group (Punjabis) with common life style

(non pesticide users), health status (normal individuals), gender (all male) and age group.

0

100

200

300

400

500

600

QQ/L

L

QQ/L

M

QQ/M

M

QR/L

L

QR/L

M

QR/M

M

RR/L

L

RR/L

M

RR/M

M

Combined Genotype

Sp

ecif

ic a

cti

vit

y (

Mean

)

Paraoxonase U/L

Arylesterase U/ml

Serum

paraoxonase

and

arylesterase

activities

lxvii

Serum arylesterase activity did not show significant differences between PON1 Q192R

genotypes (QQ, QR, RR) which indicates that serum arylesterase activity is not associated

with PON1 Q192R. However statistically significant differences between serum

arylesterase activities of individuals with PON1 LL, LM and MM genotypes indicated that

serum arylesterase activity is associated with PON1 L55M. These general trends were also

observed in most of previous studies on other populations which indicates the basic

similarities of PON1 gene expression and activity patterns among humans of different

ethnicities. However, we observed a variable distribution in allele frequencies of PON1

SNPs when we compared our data with reports from other ethnic populations around the

globe. These variations from other populations seem to be linked with ethnicity and genetic

drift with respect to geographic position of the different populations (Sanghera et al.,

1998a; Scacchi, 2003). Genetic make up of PON1 SNPs in Pakistani Punjabi population

revealed in this study was widely different from some populations, especially Japanese,

Koreans and Chinese populations. In addition some variations in serum paraoxonase and

arylesterase activities were also observed when compared with other populations.

High paraoxonase activity allele (R192 allele) shows a broad variation among

different ethnic groups. The frequency of the R192 allele generally ranged from 22% in

English population to 73% in Amazonian populations (Oleary et al., 2005; Santos et al.,

2005). Frequency of allele R192 is higher in Japanese (60%), Korean (62%), Chinese

(58%), Amazonian-Ameridiand tribes (73%) and African Beninese populations (61.2%)

(Hong et al., 2001; Scacchi et al., 2003; Santos et al., 2005; Mohamed and Chia, 2008).

The R192 allele frequency in our study was relatively low (31% in Pakistani healthy

Punjabi population) which is similar to the frequencies reported for other populations by

Sanghera et al. (1997) (33% in Indians), Sepahvand et al., 1997 (31% in Iranian), Noughera

et al., 1993 (27% in Saudi Arabia) and Bryk et al., (33% in Israel) amongst Asian

populations.

Our results of the frequency of the low-activity allele (Q192) in Pakistani Punjabi

population (69%) is consistent with Iranian (69%) Indian (67%) Israeli (67%) and

European-Brazilians (69.3%) populations. (Sanghera et al., 1997; Allerbrandt et al., 2002;

Bryk et al., 2005; Sepahvand et al., 2007). Higher frequencies are reported previously in

lxviii

some populations including Saudi Arabian (73%) and Canadian (73%) populations

(Nogueira et al., 1993; Mckeown-Eyssen et al., 2004). Frequencies in most of other

populations were lower than Pakistani Punjabi population which includes Caribean-

Hispanics (54%), Mexicans (51%), Peruvians (53.9%), Ethiopians (59.2%), Japanese

(40%), Korean (38%) and Chinese individuals (42%) (Hong et al., 2001; Chen et al., 2003;

Scacchi et al., 2003; Rojas Garcia et al., 2005; Catano et al., 2006; Mohamed and Chia,

2008).

The distribution of the L55 allele is relatively homogeneous worldwide.

Populations worldwide show predominance of L55 (74% in present study) over M55 allele

(26% in present study). Prevalence of L55 allele ranged from 96.7% for Amazonians

(Santos et al., 2005), to 59% for Iranians (Sepahvand et al., 2007). The prevalence of L55

allele in our study (74%) was higher than that reported in the Israeli (61%) (Bryk et al.,

2005) and Iranian (59%) (Sepahvand et al., 2007) populations, and lower than Japanese

(96%), Korean (94%) and Chinese (95%) inhabitants (Hong et al., 2001; Mohamed and

Chia, 2006). The occurrence of the L55 allele in our study (74%) is relatively the same as

reported in some other populations including English (70%), Turkish (70%), African

Brazilians (71.4%) and Caribian Hispanics (71%) (Oleary et al., 2005; Aynacioglu et al.,

1999; Allebrandt et al., 2002; Chen et al., 2003).

The M55 allele frequency clearly differed between various ethnic groups. The

prevalence of M55 allele in Pakistani Punjabi population (26 % M in Pakistani Punjabis)

was lower than Iranian (41%: Sepahvand et al., 2007), and Israeli (39%: Bryk et al., 2005)

populations. However, frequency of M allele was much higher in Pakistani Punjabi

population (26 % M in Pakistani Punjabis) than Japanese (4%: Mohamed and Chia, 2008),

Korean (6%: Hong et al., 2001), Chinese (5%: Mohamed and Chia, 2008) and Amazonian-

Ameridian tribes (3.3%: Santos et al., 2005).

The most common combined genotype in Pakistani Punjabi population was

QQ/LM (25%) followed by the QQ/LL (24.29%), with the low prevalence of LM/QR

(10%), RR/LL (8.57%) and RR/LM (6.43%). The least frequent combinations were

QQ/MM (3.57%), QR/MM (1.43%) and RR/MM (1.43%) which indicates a low

prevalence of these combinations in Pakistani Punjabis. A low incidence of these genotypes

lxix

was also observed previously (Phuntuwate et al. (2005) in Thai population. In some of

previous studies the MM genotype was totally absent in other populations (Santos et al.,

2005).

Serum paraoxonase and arylesterase activities are highly variable among different

individuals depending on various factors (ethnicity, PON1 SNPs, environmental and

dietary factors, etc.). Paraoxonase activities ranging from 62.6±45.8 U/L (in United

Kingdom reported by Brimohun et al., 2009) to 932.2± 528.3 U/L (in Mexico agricultural

women) has been reported (Gonzalez et al., 2012). In Pakistan, serum paraoxonase activity

has been previously reported by M. Perwaiz et al., 2007 (61±35 U/ml) and Jamall et al.,

2010 (106±19.42 U/ml) in healthy individuals without considering PON1 genotypes or

arylesterase activities. Our results indicate that the extent of arylesterase and paraoxonase

activities depends on PON1 coding region SNPs among studied population. Average value

of serum paraoxonase activity in our study was (232±75 U/L). According to respective

SNPs, the paraoxonase activity increased in the following order RR>QR>QQ and

LL>ML>MM. Highest serum paraoxonase activity was noted in RR genotype (455±57

U/L) while lowest in MM genotype (84± 49 U/L).

Average value of serum arylesterase activity in our study was (140± 59 U/ml). The

highest serum arylesterase activity (135±38 U/ml) was noted in LL genotype and the lowest

(65±42 U/ml) in MM genotype. Arylesterase activity did not show clear differences

according to Q/R polymorphism while arylesterase activity increased in the following order

with respect to L/M genotype: LL>LM>MM. Arylesterase activity was highest in QQ/LL

(140± 27 U/ml) and lowest activity was observed in QQ/MM (68±19 U/ml) and RR/LM

(80±46 U/ml). Arylesterase activity is generally less variable as compared to serum

paraoxonase activity. Equivalent values of arylesterase activities (116±33 umol/min/ml

(Eckerson et al., 1983), 132±52U/ml (Kilic et al., 2005), 83 μmol/min/ml (Mike et al.,

2005), 132.7±29.3 U/ml (Gonzalez et al., 2012)) were reported previously in other

populations.

A strong and significant association between certain genotypes of PON1 coding

region and some diseases has been reported in many of the previous studies. Once the

genotypes of a population are known, we can evaluate the population based risk of

lxx

associated diseases to at least some extent. However these predictions should be viewed

with caution as many factors may alter the ultimate outcome of PON1 activity and

concentration in humans. Here, we describe a few possible outcomes of existing PON1

SNPs of Pakistani Punjabi population studied. The Q192 allozyme was more prevalent

(69%) in Pakistani Punjabi population compared to R192 (31%) and the previous studies

suggest that Q192 hydrolyzes toxic organophosphate compounds diazoxon, sarin, soman

and phospholipids as well as cholesteryl ester hydroperoxides more rapidly as compared

to R192 allele (Davies et al., 1996; Aviram et al., 1999). The R192 allozyme hydrolyzes

methyl parathion toxic product paraoxon more efficiently than Q192 allele (Costa et al.,

2005). Thus the studied population may be more protected against toxic effects of

diazoxon, sarin, soman and cholesteryl ester hydroperoxides but less protected against

paraoxon due to their existing PON1 genotypes.

Many cancer predisposing conditions are associated with an increased sensitivity

towards chromosome damaging effects of ionizing radiations (Ron, 1998; Leeman-Neill,

2013; Santoro and Carlomagno, 2013). Although PON1 genotypes have not been studied

for any link with higher sensitivity to ionizing radiations, however, a decreased arylesterase

and paraoxonase activity was reported by Serhatlioglu et al., (2003) in irradiated workers.

This association suggested a role of PON1 in protection against toxic effects of radiation.

Thus it becomes very important to record and maintain the healthy levels of serum

paraoxonase and arylesterase activities in general population in Pakistani as well as other

populations in the present era when very unfortunately long lasting effects of radiation

accidents like Hiroshima and Nagasaki (Cullings, 2014), Chernobyl (Little et al., 2014)

and Fukoshima (Jakob et al., 2014) have emerged as a reality posing a risk of direct/indirect

exposure to radiation for every one living on earth.

Rojas-Garcia et al., 2009 investigated effect of paraoxon (a toxic OP agent) on

human lymphocytes. Cell viability of paraoxon treated human lymphocytes was not

affected by paraoxon; however a significantly increased frequency of micronucleus (MN)

was observed in QQ genotype individuals’ lymphocytes. These results indicate that QQ

genotype may be a risk factor for development of cancer due to pesticides toxicity. Our

results indicated that QQ genotype is predominant (54%) in Pakistani Punjabis. This

lxxi

important finding suggests that Pakistani Punjabi population is relatively susceptible to

development of cancer due to occupational exposure to pesticides. Indeed association of

pesticides with cancer in pesticide workers has been reported previously in the studied

Pakistani population mainly living in Punjab (Bhalli et al., 2006; Ali et al., 2008; Bhalli et

al., 2009).

If we accept that associations found between various genotypes and diseases in

previous studies are equally applicable to any of the human populations, then we can make

certain predictions about susceptibility of our studied population to various diseases on the

bases of PON1 coding region frequencies revealed in this study. We assume that these

predictions will be more accurate if other factors are kept in control to avoid their influence

on PON1 levels and activity. As PON1 192Q allele is reported to be associated with low

paraoxonase activity in humans exposed to OPs (Sunay et al., 2013) and our studied

population has higher frequency of Q allele so this population is more prone to toxic effects

of OP pesticides due to low levels of paraoxonase activity. PON1 QQ genotype and Q

allele are associated with increased risk of developing lymphohaemopoietic cancers in

agricultural communities (Kokouva et al., 2013), so Pakistani Punjabi individuals in

agricultural sector may be more prone to this type of cancer due to their PON1 genotypes

although no epidemiological studies have been conducted so far in this regard in Pakistani

Punjabis agricultural communities.

PON1 192R allele is an independent risk factor for coronary artery disease (CAD)

as indicated by a study in Irani population (Vaisi-Raygani et al., 2011). Our studied

population might be less susceptible to CAD due to low prevalence of R allele. QR and RR

genotypes are associated with higher risk of CAD (Gupta et al., 2011), so studied Punjabi

population is less susceptible to CAD because of low prevalence of these genotypes in

them. PON1 192R allele is associated with ischemic stroke (Mahrooz et al., 2012) and

frequency of this allele is low in our studied population thus only a fraction of studied

Pakistani Punjabi population may be susceptible to ischemic stroke. As PON1 192R allele

is reported to be associated with increased risk of atherosclerosis by inducing chronic low

grade inflammation (Luersen et al., 2011), so Punjabi population may be less susceptible

to atherosclerosis due to low prevalence of PON1 192R.

lxxii

PON1 55M and PON1 192R alleles are associated with decreased sperm motility

(Lazaros et al., 2011), so Punjabi population is expected to have normal sperm motility

because both PON1 55M and PON1 192R alleles are low in studied population.

PON1 192QQ genotype and PON1 55L allele are associated with a high risk of

developing osteosarcoma (Erqen et al., 2011), while both Q and L alleles have high

frequencies in Punjabi population, so they are at increased risk of developing

osteosarcoma. PON1 192RR genotype is more frequently found in coronary artery stenosis

patients of Irani population (Fallah et al., 2010) thus Punjabi population is less susceptible

to coronary artery stenosis as their PON1 192RR genotype frequency is very low.

PON1 192QQ genotype is more protective against development of bladder cancer

(Ozturk et al., 2009). Thus Punjabi population might be protected against bladder cancers.

PON1 55M allele is a risk factor for systemic lupus erythmatosus (Bahrehmand et al.,

2014) and this allele has very low frequency in Punjabis, so they are less likely to develop

systemic lupus erythmatosus. PON1 55M allele is a risk factor for cancers (Fang et al.,

2012), however frequency of this allele is low in Pakistani Punjabi population.

PON1 192QQ genotype and PON1 192Q allele is related with migraine (Garcia-

Martin et al., 2010), so studied population is at higher risk of migraine at early age. Our

prediction about migraine based on PON1 genotype is very true because Murtaza et al.,

(2009) reported that migraine is one of the most common forms of headache in Pakistanis

in their most productive ages.

It is also interesting to note that most of disease-genotype associations have

revealed significant associations of various diseases with PON1 Q192R genotypes and

associations with PON1 L55M are less reported. It may be due to the difference of

biological activity between alleles of PON1 Q192R, while allele variation of PON1 L55M

shows less difference in the catalytic activity of the PON1 protein. Population based case-

control studies have demonstrated association of a decreased serum paraoxonase and

arylesterase activities with many disease conditions including balder cancer (Aydin et al.,

2013), selective serotonin reuptake inhibitor (SSRI) intoxication (Kati et al., 2014),

organophosphate poisoning (Richard et al., 2013), Idiopathic Parkinson’s disease (Kirbas

lxxiii

et al., 2014), insomnia (Liang et al., 2013), mitral and aortic valve insufficiency (Yilmaz

et al., 2013), generalized anxiety disorder (Bulut et al., 2013), colorectal cancer (Bulbuller

et al., 2013), Sjogren’s syndrome (Szanto et al., 2010), autism (Gaita et al., 2010), obesity

(Seres et al., 2010), critical illness with sepsis (Novak et al., 2010) and chronic hepatitis

(Kilic et al., 2005). Thus establishing a normal range of serum paraoxonase and

arylesterase activities in normal populations will be helpful for diagnosis and evaluation of

prognosis/treatment of related diseases. We believe that this association of PON1 with

multiple of diseases suggests that although differential diagnosis will not be facilitated on

the basis of PON1 enzyme activity in such diseases, however prognosis and therapeutic

outcome monitoring will be facilitated by measuring improvements in serum paraoxonase

and arylesterase activities of the patients.

2.5. Conclusions

Population based studies revealed differences in the allele frequencies of PON1

gene polymorphisms and serum paraoxonase and arylesterase activities. These ethnic

differences are considerable important factors for prevention and management of various

lxxiv

diseases associated with PON1 polymorphisms and decreased PON1 enzymatic activity.

The most of results of PON1 genotypes frequencies of the current study are similar to what

has been reported for Indian, Iranian, Saudi and Israeli populations which may indicate

some common ancestral origins of these populations. The variations of PON1 genotypes

frequencies from other populations, (especially from Chinese, Korean and Japanese)

detected in this study would be due to genetic drift, geographic drift and ethnic variations.

This study provides a basic data of normal non-pesticide exposed healthy population which

will be helpful in PON1 related disease-association studies in the Pakistani population in

future. We have made certain predictions about partial susceptibility to various diseases on

the basis of our results of genotype frequencies and related disease-associations found in

previous studies. We recommend the preventive measures should be taken to protect the

studied population against these diseases. Based on genotypes, it is predicted that Punjabi

male population is more prone to paraoxon related OPs as compared to sarin, soman and

other related compounds. The reveled information will be highly valuable for general

medical practitioners and policy makers of health ministry of Pakistan and global public

health organizations including World Health Organization.

3.0 (Case study-2): Paraoxonase-1 Genotypes and Enzyme Activities in

Pakistani (Punjabi) Pesticide Sprayers: Identification of Population

Susceptible to Risk of Pesticide Toxicity

3.1. Introduction:

Extensive use of pesticides is a major public health concern of the modern era. OP

toxicity is one of the major contributes towards morbidity in developing countries (Buckley

et al., 2004). Almost one million pesticide poisoning events and twenty thousand related

lxxv

deaths occur per year in developing countries (Klein-Schwartz and Smith, 1997). Detection

of toxicity patterns and underlying susceptibility patterns will help in protecting

agricultural workers against pesticide poisoning.

Highly toxic oxon forms of OPs are hydrolyzed by PON1 (Singh et al., 2011;

Hernandez et al., 2013). Studies in animal models have indicated that PON1 is associated

with sensitivity to toxic effects of Organophosphate pesticides (OPs) (Shih et al., 1998; Li

et al., 2000; Cole et al., 2005). Decreased PON1 activity in transgenic mice leads to AChE

inhibition on exposure to oxons of chlorpyrifos and diazinone (Li et al., 2000) which

indicates protective role of PON1 against OP toxicity. Association of PON1 with

susceptibility to xenobiotics in humans have been studied in various types of exposures

including Gulf war victims, sheep dippers, household exposure and agricultural workers’

exposure to OPs (Costa et al., 2005). These studies indicate that paraoxonase is the major

polymorphic enzyme responsible for detoxification of methyl parathion and other

organophosphate pesticides (OPs) in humans in their blood and tissue fluids (Singh et al.,

2011; Mohamed and Chia 2008).

The protective function of PON1 is dependant on various factors including PON1

coding region SNPs (Singh et al., 2011). Enzymatic function of PON1 clearly defines the

level of protection against toxic effects of OPs. Previously several studies in developed

countries have shown a relation between PON1 and susceptibility to OP toxicity. However

Asian populations are less studied in this context. Ethnicity can be one of the major

contributors in susceptibility to OP toxicity because PON1 activity varies depending on

specific genotype pattern in different populations (Singh et al., 2011). Wide variations in

PON1 activity in individuals of different as well as similar ethnicity has been reported

previously (La Du et al., 1986; Furlong et al., 1988; Brophy et al., 2001; Chen et al., 2003)

which may result in predisposing low activity individuals at a greater risk of being affected

by toxic effects of OPs. This variation is linked with genotypes (homozygous or

heterozygous for low activity or fast activity allele) (Eckerson et al., 1983; Geldmacher-

von Mallinckrodt and Diepgen, 1988), resulting in higher susceptibility of low activity

allele carriers to OP toxicity which is indicated previously by animal models and

epidemiological studies (reviewed by Costa et al., 2003). Although animal studies have

lxxvi

provided a basic evidence of association of PON1 genotypes (in combination with

phenotype) with susceptibility towards OP toxicity, however, direct evidence of relevance

of PON1 status in humans in terms of susceptibility to OP toxicity is still elusive. Studies

are needed in order to document the level and consequences of exposure to OPs and their

relevance with PON1 genotypes and phenotypes at clinical levels.

Variations in PON1 genotypes and activity suggest a need to study PON1 in

populations widely exposed or at a risk of exposure to OPs for proper management of any

toxic effects. Since OP exposure leads to various types of noticeable ill health effects in

susceptible individuals, thus it is important to find out how different types of groups would

be affected with such medical problems so that medical workers and policy makers can be

properly advised to provide specific preventive or curing support to the affected

communities (Mohamed and Chia, 2008). As PON1 genotypes and enzyme activities are

different in populations of different ethnicities and localities, thus we expect to detect

specific and distinct OP susceptibility patterns by studying specific ethnic groups.

Pakistan is one of the major agricultural countries and Pakistani Punjabi population

is largely exposed to pesticides thus they are directly facing a risk of pesticide exposure.

Understanding the susceptibility and toxicity patterns will help to protect those who are at

the risk of developing toxic effects of OPs. Identification of “at-risk population” will help

to take preventive measures and improve health status of such agricultural workers. This

study was conducted in order to identify the PON1 genotypes and phenotypes of Pakistani

Punjabi population at higher risk of developing pesticide toxicity and identification of

associated risk factors and susceptibility patterns.

3.2. Materials and methods

3.2.1. Study Population

In this cross sectional study, we interviewed and sampled pesticide users (back pack

sprayers) in cotton growing areas of Pakistan (Punjab). Cotton production consumes more

than 70% of total pesticides consumed in Pakistan and at least twelve spray sessions are

applied per season (Sep to Nov.). The study was carried out at the end of the season to

cover the health effects of the whole season on pesticide sprayers. All individuals were

lxxvii

asked about their current and past disease status and their current or past pesticide exposure

status was recorded. Data was analyzed for determining prevalence of adverse health

effects in pesticide users alongwith any differences between lifestyle, pesticide

consumption, safety measures, PON1 genotypes and serum paraoxonase and serum

arylesterase activities. Total number of sampled pesticide users reporting toxic effects of

pesticides was 101 and total number of pesticide users without any toxic effects of

pesticides was 100. These pesticide users were involved in all the activities including

handling, mixing and spraying of the pesticides.

3.2.2. Methods

Methods were same as described in previous section (case-1) (Chapter 2.0; pages 16-19).

In addition structural analysis of PON1 SNPS was done by Modelling PON1 protein

structures and docking. Structure file of the human PON1 1VO4 was retreived from PDB

database, the structure has been generated through crystallizing a genetically engineered

protein with a number of mutations. Native PON1 structure and those of L/M 55 And R/Q

192 SNPs were modelled at modller software. Therefore four models for PON1 M55,

Q192, L55 and R192 SNPs were generated. All the models were validated on the basis of

various statndard thresholds such as z-score and phi/psi plots.

3.3. Results

3.3.1. Study population

There were no statistically significant differences between age, gender, BMI,

ethnicity and method of pesticide use of affected and healthy pesticide sprayers. Tobacco

consumption was significantly higher in affected persons (P<0.05) as compared to healthy

lxxviii

pesticide workers. There were no significant differences in alcohol consumption while

alcohol consumption was very low in both groups (Table 3.1).

Table 3.1: Demographic features of Pakistani Punjabi Pesticide workers

Features Affected (n = 101) Normal (n = 100) P values

Age 32±19 29±15 NS

Male 101 100 NS

Female 0 0 NS

Smoker 47 24 P<0.05

Alcohol consumer 2 0 NS

NS: Statistically non significant difference between affected and normal pesticide workers

3.3.2. Types of pesticides used

No significant differences of types of pesticide use (Organophosphates,

Carbamates, Organochlorines, Pyrethroids) were observed between healthy and affected

pesticide workers (Fig 3.1). All the participants had used organophosphate pesticides

(100%) because non-OP users were excluded from study during sampling. Use of

Organochlorines was low (30% and 32% in affected and normal pesticide users) as

lxxix

compared to other pesticides. Increasing order of pesticide use was as follows:

Organophosphates> Pyrethroids>Carbamates>Organochlorines.

Fig. 3.1: Comparison of pesticide usage in studied Pakistani Punjabi Pesticide workers.

NS: statistically non-significant differences between affected (n = 101) and non-affected

(n = 100) pesticide users

3.3.3. Personal Protective Equipments (PPEs)

Significant differences (P<0.05) were observed for use of personal protective

equipments (eye glasses, cap, head cover, Long shoes, Boots, Face mask, Romaal (a

50%

100%

30%

60%56%

100%

32%

78%

0%

20%

40%

60%

80%

100%

120%

Carbamates Organophosphates Organochlorines Pyrethroids

Pre

va

len

ce

Affected

Normal

NS

NS

NS

NS

lxxx

traditional face and head cover) and Gloves) between the affected and non- affected

individuals. Use of standard modern PPEs was lacking in both the groups of pesticide

workers (Fig 3.2).

Fig. 3.2: Comparison of personal protective equipment (PPE) usage in studied Pakistani

Punjabi Pesticide workers (back pack sprayers). *: statistically significant differences

(P<0.05) between affected (n = 101) and non-affected (n =100) pesticide users

3.3.4. Safety Precautions about wind direction

10%

40%

50%

10%

56%

5%

30%

70%

80%

35%

78%

16%

0% 20% 40% 60% 80% 100%

Gloves

Mask/Romal

Boots/Closed

shoes

Long shoes

Cap/ head

cover

Eye Glasses

Prevalence

Normal

Affected

*

*

*

*

*

*

lxxxi

Majority of the pesticide workers (55% affected and 33% non-affected workers)

did not pay any attention about direction of wind during pesticide spraying. Significantly

lower number of affected workers (20%) had sprayed in correct wind direction (in the same

direction in which air flowed) as compared to non affected pesticide workers (40%) (Fig

3.3).

Fig. 3.3: Comparison of safety measure about wind direction in studied Pakistani Punjabi

Pesticide workers (back pack sprayers). NS: statistically non-significant differences

between affected and non-affected pesticide users. *: statistically significant differences

(P<0.05) between affected (n=101) and non-affected (n=100) pesticide users.

20%

25%

55%

40%

27%

33%

0% 10% 20% 30% 40% 50% 60% 70%

Sprayed in wind

direction

Sprayed

against wind

direction

No attention to

wind direction

Normal

Affected

*

*

NS

lxxxii

3.3.5. Safety precautions after working with pesticides

Safety precautions after working with pesticides (washing hands, face and feet,

mouth wash, body wash, change of clothes) were significantly low in affected pesticide

workers (P<0.05) as compared to healthy sprayers (Fig 3.4).

Fig. 3.4: Comparison of safety measures (after pesticide spraying) in studied Pakistani

Punjabi Pesticide workers (back pack sprayers). NS: statistically non-significant

differences between affected and non-affected pesticide users. *: statistically significant

differences (P<0.05) between affected (n=101) and non-affected (n=100) pesticide users.

70%

20%

5%

10%

89%

60%

10%

15%

0% 20% 40% 60% 80% 100%

Hand wash

Mouth wash

Body wash

Clothes

changed

Normal

Affected

*

*

NS

NS

lxxxiii

3.3.6. Sources of training and guidance

No significant differences were present in source of the pesticide usage training of

both groups of pesticide workers and both of the groups lacked appropriate training of

pesticide use (Fig 3.5). Majority of pesticide users (90% normal and 93% affected) were

trained by their co-workers. None of them (0%) was trained by pesticide manufacturers or

authorized government agencies.

Fig. 3.5: Comparison of sources of training in Pakistani Punjabi Pesticide workers (back

pack sprayers). NS: statistically non-significant differences between affected and non-

affected pesticide users. *: statistically significant differences (P<0.05) between affected

(n = 101) and non-affected (100) pesticide users.

0%

0%

7%

93%

0%

0%

10%

90%

0% 20% 40% 60% 80% 100%

From

government

agencies

From pesticide

manufacturers

From pesticide

sellers

From co-

workers

Normal

Affected

NS

NS Pesticide

Usage

Training

Received

lxxxiv

3.3.7. Direct body contact with pesticides during work

Different body parts of the affected pesticide workers (feet, face, legs, arms and

hands) were significantly highly exposed to direct contact with pesticides in affected

pesticide workers (P<0.05) as compared to non affected pesticide workers (Fig 3.6). Hands

were the most frequently exposed body parts (70% affected and 50% non affected pesticide

users) while legs were the least frequently exposed parts (2% in normal and 10% in affected

pesticide users).

Fig. 3.6: Comparison of direct body contact (direct exposure) with pesticides in studied

Pakistani Punjabi Pesticide workers (back pack sprayers). NS: statistically non-significant

differences between affected and non-affected pesticide users. *: statistically significant

differences (P<0.05) between affected (n = 101) and non-affected (n = 100) pesticide users.

70%

60%

10%

20%

30%

50%

40%

2%

4%

9%

0% 20% 40% 60% 80%

Hands

Arms

Legs

Face

Feet

Normal

Affected

*

*

*

*

*

lxxxv

3.3.8. Signs and symptoms of acute toxicity recorded in affected individuals

The signs and symptoms of pesticide toxicity observed in affected individuals

included headache, nausea, dizziness, vomiting, muscle weakness, difficulty in conceiving

(mainly related to nervous system indicating absorption into blood), rhinitis, dyspnoea,

bronchitis, asthma, (related to respiratory system effects), dermatitis and allergy (related to

skin exposure) (Fig 3.7).

64%

57%

19%

12%

35%

49%

28%

30%

7%

19%

16%

20%

0% 20% 40% 60% 80%

Allergy

Dermatitis

Difficulty in conceiving

Asthma

Bronchitis

Dyspnoea

Rhinitis

Muscle weakness

Vomiting

Dizziness

Nausea

Headache

Prevalence

lxxxvi

Fig. 3.7: Signs and symptoms of acute toxic effects of organophosphate pesticides in

affected pesticide workers (n = 101)

3.3.9. PON1 SNPs

Prevalence of PON1 coding region alleles showed three different patterns. PON1

192R (51.5%) and PON1 55L alleles (81.5%) were dominant in normal pesticide workers

(Table 3.2). PON1 192Q (69.31%) and PON1 55M alleles (52.14) were predominant in

affected pesticide workers (Table 3.3). PON1 192Q (68.93%) and PON1 55L allele

(73.93%) were dominant in healthy non-pesticide exposed Punjabis (Table 3.4).

The prevalence of low activity (PON1 192Q) allele (69.31%) was significantly

higher in affected workers compared with normal pesticide workers (48.5%) (Table 3.4).

Low expression (PON1 55M) alleles (52.14%) was significantly higher in affected workers

(P<0.05) as compared to healthy workers (18.5%) and non-pesticide exposed healthy

Punjabis (26.07%) (Table 3.4).

PON1 192QQ genotype was significantly higher (52.48%) in affected workers

compared with normal workers (30%). PON1 192RR genotype was significantly lower

(14.85%) in affected workers compared with normal workers (33%). PON1 55LL genotype

was significantly lower in affected workers (28.71%) compared with healthy workers

(54%) and healthy non-pesticide exposed Punjabis (52.86%). PON1 55MM genotype was

significantly high (34.66%) in affected workers compared with healthy workers (2%) and

non-pesticide exposed Punjabis (5%) (Table 3.4).

QQ/MM genotype was higher (13.86%) in affected workers compared with healthy

workers (0%) and non-pesticide exposed healthy Punjabis (3.57%). QR/MM genotype was

also higher in affected pesticide workers (14.85%) compared to healthy workers (2%) and

lxxxvii

non-pesticide exposed Punjabis (1.43%). RR/LL genotype in affected workers (5.94) was

significantly lower in affected workers than healthy workers (23%) (Table 3.4). Graphic

(3D) Presentation of combined genotypes of PON1 Q192R and PON1 L55M highlighted

three different patterns of PON1 genotypes in affected pesticide workers, non affected

pesticide workers and healthy Punjabi population (Fig 3.8).


in healthy Pakistani Punjabi pesticide sprayers (n=100).

Polymorphism Allele

n = 200

Frequency Genotype

n = 100

Frequency

N % N %

PON1 Q192R Q

R

97

103

48.5

51.5

QQ

QR

RR

30

37

33

30

37

33

PON1 L55M L

M

163

37

81.5

18.5

LL

LM

MM

64

34

2

64

34

2


in affected Pakistani Punjabi pesticide sprayers (n=101).

Polymorphism Allele

n = 202

Frequency Genotype

n = 101

Frequency

N % N %

lxxxviii

PON1 Q192R Q

R

140

62

69.31

30.69

QQ

QR

RR

53

33

15

52.48

32.67

14.85

PON1 L55M L

M

96

104

47.85

52.14

LL

LM

MM

29

38

34

28.71

37.62

33.66

Table 3.4: Comparison of PON1 genotypes between controls and pesticide workers

Genotype Punjabi healthy

males n (%) (n=140)

Healthy sprayers

n (%) (n=100)

Affected sprayers

n (%) (n=101)

Q

R

L

M

QQ

QR

RR

LL

LM

193 (68.93%)

87 (31.07%)

207 (73.93%)

73 (26.07%)

76 (54.29%)

43 (30.71%)

21 (15%)

74 (52.86%)

59 (42.14%)

97 (48.5%)

103 (51.5%)

163 (81.5%)

37 (18.5%)

30 (30%)

37 (37%)

33 (33%)

64 (64%)

34 (34%)

140 (69.31%) b

62 (30.69) b

96 (47.85%) a, b

104 (52.14) a, b

53 (52.48%) b

33 (32.67%)

15 (14.85%) b

29 (28.71%) a, b

38 (37.62%)

lxxxix

MM

QQ/LL

QQ/LM

QQ/MM

QR/LL

QR/LM

QR/MM

RR/LL

RR/LM

RR/MM

7 (5%)

34 (24.29%)

35 (25%)

5 (3.57%)

27 (19.29%)

14 (10%)

2 (1.43%)

12 (8.57%)

9 (6.43%)

2 (1.43%)

2 (2%)

15 (15%)

15 (15%)

0 (0%)

26 (26%)

9 (9%)

2 (2%)

23 (23%)

10 (10%)

0 (0%)

34 (34.66%) a, b

19 (18.81%)

21 (20.79%)

14 (13.86%) a, b

4 (3.96%) a, b

13 (12.87%)

15 (14.85%) a, b

6 (5.94)% b

4 (4.04%)

4 (4.04%)

a: Significant difference (P<0.05) compared with healthy non-pesticide worker Punjabis;

b: Significant difference (P<0.05) compared with healthy pesticide workers

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

Punjabi healthy

population (n = 140)

xc

Fig. 3.8: Comparison of genotypes between affected (n = 101) and non affected (n = 100)

spray workers and healthy Punjabis (n = 140).


The arylesterase activity was significantly higher (P<0.05) in non-affected pesticide

workers as compared to affected workers in all the genotypes of PON1 Q192R and PON1

L55M. On the other hand the values of serum arylesterase activity in affected workers were

significantly low (P<0.05) for all genotypes except MM genotype when compared with

normal healthy non exposed Punjabi population. Serum arylesterase activity of affected

pesticide workers ranged from 50±11 U/mL for QQ genotype to 87± 22 U/mL for LL

LL LM MM RR Q

R QQ

0

5

10

15

20

25

30

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

LL LM MM RR Q

R QQ

0

2

4

6

8

10

12

14

16

18

20

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

Punjabi healthy

pesticide sprayers

(n = 100)

Punjabi affected

pesticide sprayers

(n = 101)

xci

genotype while serum arylesterase activity of non affected pesticide workers ranged from

80± 21 U/mL to 141±.39 U/ml for RR genotype.

Fig. 3.9: Differences of serum arylesterase activities in affected pesticide sprayers (n =

101) compared with non-affected pesticide workers (n = 100) and healthy non pesticide

exposed Punjabi population (n = 140). a: statistically significant difference (P<0.05)

between affected sprayers and non affected sprayers. b: statistically significant difference

(P<0.05) between affected pesticide workers and normal healthy non-pesticide exposed

Punjabis


Serum paraoxonase activity (U/L) was significantly lower (P<0.05) in affected

pesticide workers (94±11 U/L for QQ genotype to 301±58 U/L for RR genotype) as

compared to non-affected pesticide workers (159±18 U/L for QQ genotype to 491±68 U/L

for RR genotype) as well as when compared with healthy non-pesticide exposed Punjabis

0

20

40

60

80

100

120

140

160

180

200

QQ QR RR MM LM LL

PON1 genotypes

Seru

m A

ryle

ste

rase a

cti

vit

y

(U/m

L)

Non affected sprayers Affected sprayers Healthy Punjabis

a, b

a, b

a, b

a

a, b

a, b

xcii

in QQ, QR, RR, LM and LL genotypes. Serum paraoxonase activity (74±10 U/L) was

lower in affected pesticide workers as compared to non-affected pesticide workers (100±19

U/L) as well as when compared with healthy non-pesticide exposed Punjabis in MM

genotypes also, however the difference was not significant.

Fig. 3.10: Differences of serum paraoxonase activities (U/L) in affected pesticide sprayers

(n = 101) compared with non-affected pesticide workers (n = 100) and healthy non

pesticide exposed Punjabi population (n = 140). a: statistically significant difference

(P<0.05) between affected sprayers and non affected sprayers. b: statistically significant

difference (P<0.05) between affected pesticide workers and normal healthy non-pesticide

exposed Punjabis. NS: non significant differences.

3.3.12. Serum arylesterase activities according to combined genotypes

Serum arylesterase activities (U/mL) were significantly low in affected pesticide

workers (P<0.05) as compared to non affected pesticide workers and non-pesticide exposed

-50

50

150

250

350

450

550

650

QQ QR RR MM LM LL

PON1 genotypes

Seru

m P

ara

oxo

nase a

cti

vit

y

(U/L

)

Series1 Series2 Series3

a, b

a, b

a, b

NS

a, b

a, b

xciii

healthy Punjabis with respect to combined genotypes. However differences for QQ/MM

and RR/MM genotypes were not significant.

Fig. 3.11: Comparison of serum arylesterase activity (U/mL) in different genotypes of

affected pesticide sprayers (n = 101) compared with non-affected sprayers (n = 100) and

non- exposed healthy Punjabi population (n = 140). a: statistically significant difference

(P<0.05) between affected sprayers and non affected sprayers. b: statistically significant

difference (P<0.05) between affected pesticide workers and normal healthy non-pesticide

exposed Punjabis. NS: non significant differences.

3.3.13. Serum paraoxonase activities according to combined genotypes

0

50

100

150

200

250

300Q

Q/L

L

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM

PON1 combined genotype

Seru

m a

ryle

ste

rase a

cti

vit

y

Non-affected sprayers Affected sprayers Healthy Punjabis

a, b

a, b

NS

a, b

a, b

a, b

a, b

a, b

NS

xciv

Serum paraoxonase activities (U/L) were significantly low in affected pesticide

workers (P<0.05) as compared to non affected pesticide workers as well as compared to

healthy non-pesticide exposed healthy Punjabis with respect to combined genotypes.

Fig. 3.12: Differences of serum paraoxonase activities (U/L) in affected pesticide sprayers

(n=101) compared with non-affected pesticide workers (n=100) and healthy non pesticide

exposed Punjabi population (n = 140) with respect of combined genotypes of PON1 Q192R

and PON1 L55M. a: statistically significant difference (P<0.05) between affected sprayers

and non affected sprayers. b: statistically significant difference (P<0.05) between affected

pesticide workers and normal healthy non-pesticide exposed Punjabis. NS: non significant

differences.

-50

50

150

250

350

450

550

650Q

Q/L

L

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM

PON1 combined genotypes

Seru

m P

ara

oxo

nase a

cti

vit

ies

Non-affected sprayers Affected sprayers Healthy Punjabis

a, b

a, b

a, b

a, b

a, b

a, b

a, b

a, b

a

xcv

3.3.13. Structural Modeling of PON1 protein

Methyl parathion was docked in the PON1 protein models according to SNPs. The models

docked with MP at Fig. 3.13 and 3.14 revealed that the binding pocket is marginally dilated

in case of PON1 R192 as compared to that of Q192 structures. The volume of PON1-Q192

binding pocket is significantly lesser thant that of PON1-R192. With a narrower binding

pocket the affinity of methyl parathion gets compromised thereby affecting paraoxonase

and arylesterase activities of the protein. Hinder to the MP binding pocket lie two calciaum

ions, the ions are stabilised by by the polar residues such as ASP 54, ASP169, ASN224

and ASN 267, L55 also lies closer the Ca2+ ions in the close proximity of the binding

pocket, Fig. 3.15. In case of L/M55 PON1 the Ca2+ part of the PON1 ninding pocket gets

further charged inducing stronger interaction, constricting the binding pockect further and

compromising the MP binding at PON1 affecting the paraoxonase and arylesterase

activities of the protein.

Fig. 3.13: Methyl Parathion (MP) docked at PON1 (R192) binding pocket. Left: MP

binding pocket surface shown in pink and blue. Arginine 192 conforms away from the

binding pocket rendering the overall binding pocket volume dilated facilitating MP

catalysis with minimum steric hindrance. Right: the same as left without surface, notice

R192 distant enough the MP docked inside the pocket, causing the least steric and chemical

hindrance for MP’s terminal -NO arms thereby facilitating binding and arylesterase

activity of the enzyme.

xcvi

Fig. 3.14: Methyl Parathion (MP) docked at PON1 (Q192) binding pocket. Left: MP

binding pocket surface shown in pink and blue. Glutamine 192 conforms right into binding

pocket narrowing the MP entry and the overall binding pocket volume thereby hindering

MP access to the catalytic site, causing steric hindrance for large MP molecule. Right: the

same as left without surface, notice Q192 near the MP docked inside the pocket, causing

sterically difficult for MP binding as well as repelling its terminal -NO arms.

xcvii

Fig. 3.15: Methyl Parathion (MP) docked at PON1 binding pocket. Left: MP binding at

PON1 (M55) pocket, negatively charged M would interact with Ca2+ Ions, shown with

the red arrows, thereby further constricting the binding pocket, rendering it difficult for MP

to bind, compromising the esterase activity of the enzyme . Right: MP binding at PON1

(L55) pocket, L has a little affinity for Ca2+ Ions, thereby binding pocket remains dilated

enough for MP binding facilitating aryl esterase and paraoxonase activity.

xcviii

3.4. Discussion:

This is one of the first study which addressed the susceptibility and protection levels

of pesticide sprayers of Pakistan in the terms of PON1 SNPs and its enzymatic activity

along with some of non-genetic factors and signs and symptoms of toxic effects of

pesticides. The pesticide users included in this study were intensively engaged in

agricultural activity with more than five years of exposure to pesticides. They were

routinely engaged in pesticide spraying activity on weekly basis. None of them were blood

relatives of each other, except that all were of Punjabi ethnicity.

Humans are highly prone to toxic effects of pesticides. Chronic toxic effects of OPs

are reported previously by several studies in various populations (Recio et al., 2001;

Levario-Carrillo et al., 2004; Sanchez-Pena et al., 2004). Ahmad et al., (2002) has

previously reported a death rate of 64% associated with pesticide poisoning events in

Multan (Punjab, Pakistan). Afzal et al., (2006) has reported a rate of 21 percent poisoning

cases related to pesticides in hospitalized patients of the same region (Punjab, Pakistan)

(Afzal et al., 2006). It is reported in various studies that prolonged use of pesticides is

associated with gross anomalies including cancers of various organs and immunological

problems (Cabello et al. 2001; Daniel et al. 2002; Peres et al. 2006; Merhi et al. 2007). A

study in Vietnami agricultural workers revealed fifty four poisoning events per month in a

small group of 54 participants while only 2 cases per month were treated at clinics (Murphy

et al., 2002). A study in Indonesian pesticide sprayers indicated >3 symptoms of OP

toxicity in 21% individuals (Kishi et al., 1995). Three percent of agricultural workers in

developing countries (equivalent to a population of 25 million individuals out of 830

million agricultural workers) suffer one event of pesticide poisoning per year. These figures

indicate extensive us of OPs in developing countries (Asia and Africa) (WHO 1990;

Mohamed and Chia 2008). However signs and symptoms of mild and moderate acute toxic

effects of pesticides are far less studied as compared to chronic toxic effects. Thus mild

and moderate symptoms associated with low exposure of OP toxicity are mostly neglected.

A significantly decreased paraoxonase activity without any noticeable health effects was

xcix

reported by Zhang et al., 2014 in Chinese Han population repeatedly exposed to OP

pesticides. Allele 55M and R192 were predominant in Han population. In our population

allele 55M and 192Q were predominant in affected pesticide workers and we observed

acute symptoms of OP toxicity in affected pesticide sprayers in the following order: allergic

reaction (64%), dermatitis (57%), dyspnoea (49%), bronchitis (35%), muscle weakness

(30%), rhinitis (28%), headache (20%), dizziness (19%), difficulty in conceiving (19%),

nausea (16%), asthma (12%) and vomiting (7%). This profile of signs and symptoms may

be helpful for local clinicians for proper diagnosis of suspected cases of pesticide toxicity.

These symptoms are mainly related to cholinergic crisis (Namba et al., 1971; Mohamed

and Chia, 2008) and less related to organophosphate induced delayed polyneuropathy

(OPIDP) (Jokanovic et al., 2002). Protection against toxic effects of OPs is affected by

PON1 genotypes. PON1 192R allele carriers are more protected against toxic effects of

OPs related to paraoxon (Humbert et al., 1993) due to faster effect of 192R in hydrolyzing

paraoxon. Other OPs (diazoxon, etc.) are hydrolysed faster by 192Q allele (Davies et al.,

1996; Li et al., 2000). Phenylacetate is hydrolysed at similar rate by both allozyme (La Du

et al., 1986). As Q allele is predominant in affected Punjabi pesticide workers (69.31%) as

compared to non affected workers (48.5%), so toxic effects observed in this study may be

related mainly with paraoxon related pesticides. We can not exclude the possibility of

effects of other pesticides as the workers had also used other pesticides (Carbamates,

Organochlorines and Pyrethroids) in addition to OPs. However the observed signs and

symptoms are mainly related with cholinergic crises so we can safely conclude that the

observed toxic effects are mainly related to paraoxon related pesticides which is related

with high frequency of Q allele in affected pesticide workers.

Knowing the “susceptibility pattern of pesticide workers” will help to protect them

against toxic effects and ultimately improve the agricultural production and economy.

Despite the differences between different populations, finding out some common trends in

genotypes/ phenotypes and susceptibility patterns across different populations would be

very beneficial for establishing generalized rules applicable globally to all pesticide users

of all the ethnicities. Variations in paraoxonase activity and genotoxicity are associated

with PON1 coding region polymorphism, indicating the importance of PON1 coding

region polymorphism in identifying individuals at higher risk of OP toxic effects (Singh et

c

al., 2011). Most of previous studies have related PON1 Q192R with susceptibility to OP

toxicity, while our results additionally highlight the role of PON1 L55M and some of the

non-genetic factors. PON1 55M allele is rare in some populations including Chinese

individuals (Sanghera et al. 1998a, b; Wang et al. 2003; Zhang et al. 2006). PON1 55M

allele was predominant in affected Pakistani Punjabi pesticide users (52.14%) compared

with healthy pesticide workers (18.5%) and healthy non-pesticide exposed Punjabis

(26.07%). Thus PON1 55M allele (low expression allele) may be responsible for low serum

concentration of PON1 in affected individuals (indicated by low serum arylesterase

activities) in affected pesticide users as compared to healthy pesticide users and normal

non-pesticide exposed healthy Punjabis.

High activity allele (RR genotype) is predominant in Nigerian population while

Japanese and Chinese have predominance of intermediate activity allele (QR genotype)

and British, German, Sweden, Italian have low activity allele (QQ genotype) in

predominance (Enders and Mallinckrodt, 1980; Lin et al., 2002). In our study, QQ

genotype (low paraoxonase activity genotype) was predominant (52.48%) in affected

Punjabi pesticide workers and non-pesticide exposed Punjabis (52.48%). However QQ

genotype was significantly (P<0.05) low in healthy pesticide workers (30%) compared

with affected workers (52.48%) and non-pesticide exposed Punjabis (54.29%). Low

prevalence of QQ genotype (low paraoxonase activity genotype) might have been

associated with protection of non-affected workers against toxic effects of OPs in our

study.

Results of the previous studies indicate that paraoxonase enzyme activity is a better

marker of susceptibility as compared to genotypes alone because level of expression may

be altered by additional factors (environmental and dietary factors) and it is the ultimate

enzymatic activity which is responsible for protective function. Serum paraoxonase and

arylesterase activities in affected Pakistani Punjabi pesticide workers were significantly

lower than healthy pesticide users and healthy non-pesticide exposed Punjabis.

Significantly higher levels of paraoxonase activity has also been reported in several

previous studies in exposed individuals as compared to non-exposed individuals

(Mohamed and Chia, 2008) which indicates upregulation of PON1 activity in pesticide

ci

exposed individuals. Hernandez et al., 2013, showed that long term exposure to OP

pesticides increased paraoxonase activity. Our results indicate that serum paraoxonase

activity varies between various PON1 genotypes. A significant association (P<0.05) of

paraoxonase activity and Q192R genotype was also reported in pesticide workers as well

as non exposed control group by Suny et al., (2003). Our results indicate that R192 allele

carriers (QR and RR genotype) had higher paraoxonase activity while R192 deficient (QQ

genotype) individuals showed a lower paraoxonase activity which is in accordance with

results of Sunay et al., (2013).

Lee et al., 2013 indicated that either LM heterozygous genotype or QR

heterozygous genotype may be sufficient for identifying individuals with higher

susceptibility to toxic effects of OPs. Frequency of these susceptibility predictive

genotypes was not much different between affected and non affected Punjabis in our study.

Frequency of LM was 37.62%, 34% and 42 % while frequency of QR heterozygous

genotype was 32.67%, 37%, 30.71% in affected Punjabi workers, in non affected Punjabi

workers and non-pesticide exposed Punjabis respectively. However, frequency of MM

homozygous genotype was significantly high (P<0.05) (34.66%) in affected workers

compared to 2% in healthy workers and 5% in non-pesticide exposed Punjabis. This

significant difference in occurrence of MM genotype might have contributed to toxic

effects in affected pesticide workers because MM genotype (both alleles being “slow

metabolizer”) is the most susceptible genotype for OP toxic effects (Lee et al., 2013). In

addition, Fast metabolizer genotype and LL was significantly low (28.71%) in affected

workers compared with non-affected workers (54%) and non-pesticide exposed Punjabis

(52.86%). Both L55M and Q192R have significant effect on paraoxonase activity (Ellison

et al., 2012). Highest paraoxonase activity with respect to L55M was detected in LL

(301±64 U/L in non affected workers, 271±51 U/L in non-pesticide exposed Punjabis and

149±26 U/L in affected workers) and lowest (67±9 U/L in non-pesticide exposed Punjabis,

59±10 U/L in affected workers, 101±12 U/L in non-affected workers) in MM genotype

(P<0.05) in our study. These results are in accordance with the results of Singh et al., (2011)

and Ellison et al., (2012).

cii

A decreased level of arylesterase (along with decrease in total leukocyte count and

higher number of lymphocytes) is recently reported by Bernal-Hernandez et al., (2014) in

farm workers compared with non-exposed healthy controls. Serum arylesterase activities

of affected pesticide workers in all genotypes in our study were significantly lower

(P<0.05) in affected pesticide workers as compared to non-exposed Punjabis and normal

workers (except MM, QQ/MM and RR/MM individuals in which differences were not

significant). Arylesterase activity was significantly affected by PON1 genotypes in

Pakistani Punjabis (RR>QR>QQ and LL>LM>MM), which is in accordance with

Hernandez et al., (2013) and other studies which showed that PON1 Q192R (especially

RR genotype) significantly affected arylesterase activity. On the other hand, no effect of

PON1 genotype and phenotype was observed on arylesterase activity by Singh et al.,

(2011).

Taken together our results of PON1 genotypes and phenotypes indicate that

pesticide workers with higher frequency of M and Q alleles and lower serum paraoxonase

and arylesterase activities are more prone to develop pesticide toxic effects. In addition

serum paraoxonase and arylesterase activities in pesticide users as well as healthy Punjabis

are dependant on PON1 genotypes. Thus allowing only individuals with more protective

genotypes and maintaining adequate levels of paraoxonase and arylesterase activities (with

diet, and other non-genetic factors) will be helpful in reducing toxic effects in pesticide

workers.

In addition to PON1 genotype and phenotype, we also addressed some non genetic

factors possibly affecting susceptibility of pesticide workers to toxic effects. Studies

addressing non-genetic factors in addition to PON1 genotype and activity would be

beneficial in understanding the effects of gene-environment interactions on pesticide

workers’ health status (Wallace et al. 2000). Non-genetic factors including tobacco

smoking and alcohol consumption are known to affect PON1 activity (Adkins et al. 1993;

Humbert et al. 1993; Primo-Parmo et al. 1996; James et al. 2000; Kujiraoka et al. 2000;

Cherry et al. 2002; Lee et al. 2003; Rao et al. 2003). Tobacco consumption was

significantly high in affected pesticide sprayers (46.53%) as compared to non-affected

sprayers (24%) which may have partly contributed in decreasing paraoxonase activity in

ciii

affected pesticide sprayers. However alcohol consumption was very low in affected

(1.98%) as well as healthy pesticide workers (0%) and the difference was non significant.

Poor health status in pesticide workers is associated with chronic exposure to OPs

(Hernandez et al., 2005; Lopez et al., 2007; Catano et al., 2008). Pesticide poisoning is

associated with more deaths as compared to infectious diseases due to low living standards

of health safety in developing countries (Shou-zhen et al., 1987; Jeyaratnam, 1990;

McCauley et al., 2006). On the other hand, total mortality of pesticide workers is not

grossly higher as compared to normal non-exposed individuals in developed countries due

to a healthier lifestyle of their pesticide workers (Maroni and Fait, 1993: literature review

of studies between 1975 and 1991). Thus it is suggested that the health status of affected

individuals can be improved by promoting a healthier lifestyle in them. Our results indicate

that majority of Pakistani Punjabi pesticide users do not practice healthy lifestyle and they

are working without proper training and guidance and without adequate personal protective

equipments (PPE). This aspect is not given due importance in the previous studies. Only a

few studies have discussed the risk of pesticide toxicity related to safety measures and use

of PPEs. Use of standard PPEs was negligible in affected as well as non affected Punjabi

pesticide workers in the current study. Use of only a few protective equipments (eye

glasses, cap/head cover, long shoes, boots/closed shoes, mask/Romaal and gloves) was

reported by Punjabi pesticide workers in current study which was significantly low in

affected pesticide workers as compared to non-affected workers. Pesticide workers with

little or no access and knowledge about standard operating procedures, PPEs and safety

precautions are more widely exposed to pesticides (Pimetal et al., 1992) which leads to

increased pesticide residue levels in blood of these individuals (Ansari et al., 1997; Ahad

et al., 2006) leading to toxic effects. In one recent study on use of PPEs in Egyptian

pesticide workers, (Lein et al., 2012) relatively high exposure (leading to higher risk of

toxicity) was reported in fraction of participants who did not use standardized equipments

and standardized operating procedures. Thus it is concluded from our results combined

with literature that low use of PPEs by Punjabi pesticide workers is also one of the risk

factors for pesticide toxicity.

civ

In addition, Pakistani Punjabi pesticide workers with ill health had sprayed mainly

without considering wind direction (55%) as compared to non-affected pesticide sprayers

(33%). This may have contributed to higher exposure of affected workers to pesticides

adding risk of pesticide toxicity. General safety measures after spraying pesticides

(changing clothes, hand wash, mouth wash, body wash) was also significantly low in

affected pesticide users as compared to non affected pesticide users. Feet, face, legs, arms

and hands were the main body surfaces which came in direct contact with pesticides while

working. Significantly higher number of affected pesticide users reported direct contact

with pesticides as compared to non-affected pesticide users. None of the pesticide users

has been properly trained by any government agency or pesticide manufacturers. Majority

of them (93% of affected and 90% of non-affected pesticide sprayers) followed the

instructions of their co-workers (who are also not trained themselves) for pesticide use

while 7% of affected and 10% of non-affected pesticide users followed the guidance of

local pesticide sellers. These results indicate lack of proper training of pesticide users in

Pakistan which may ultimately lead to malpractice and higher exposure to pesticides

leading to toxic effects.

In addition, PON1 protein modeling revealed that the volume of PON1-Q192 binding

pocket is significantly lesser thant that of PON1-R192. With a narrower binding pocket the

affinity of methyl parathion gets compromised thereby affecting paraoxonase and

arylesterase activities of the protein. Hinder to the MP binding pocket lie two calciaum

ions, the ions are stabilised by by the polar residues such as ASP 54, ASP169, ASN224

and ASN 267, L55 also lies closer the Ca2+ ions in the close proximity of the binding

pocket, Fig. 3.15. In case of L/M55 PON1 the Ca2+ part of the PON1 ninding pocket gets

further charged inducing stronger interaction, constricting the binding pockect further and

compromising the MP binding at PON1 affecting the paraoxonase and arylesterase

activities of the protein. These results indicate that PON1 genotypes affect catalytic activity

of PON1 protein through structural changes in the PON1 protein.

cv

3.5. Conclusions:

Our results indicate significant differences of PON1 genotypes and some of non-

genetic factors between affected and normal sprayers. Pesticide users reporting toxic

effects related to OPs/ pesticides are carriers of less protective alleles and genotypes, lower

paraoxonase and arylesterase activities and low level of safety precautions. Management

of controllable risk factors may increase the health status of otherwise susceptible pesticide

users. The study highlights a gross need to train pesticide workers for proper use of

protective equipment and standard operating procedures in order to minimize their

exposure to pesticides. In addition, all pesticide users must be properly screened for their

PON1 genotype and phenotypes and level of training before allowing them to work as a

pesticide worker in order to prevent toxic effects in pesticide workers.

cvi

4.0 Serum Paraoxonase and Arylesterase Activity in Familial

Hypercholesterolemia Patients with Mutated LDLR Gene

4.1. Introduction

Human paraoxonase is primarily considered as an enzyme which protects against

the toxic effects of the organophosphate pesticides. However its role has also been studied

in other physiological processes including protection against atherosclerosis. PON1

contributes to antioxidant and anti-atherosclerotic properties of HDL-C (Yilmaz, 2012;

Eren et al., 2013). “Healthy HDL” particles contain active PON1 and suppress the

formation of oxidized lipids while “Dysfunctional HDL” particles have reduced PON1

enzyme activity which leads to greater formation of oxidized lipids and lipoproteins paving

the way to atherosclerosis (Eren et al., 2013). Gugliucci et al., (2014) has suggested the

role of PON1 associated with small dense low density lipoprotein (sdLDL) as defense

mechanism against oxidation, thus protecting against atherosclerosis. Several studies have

shown that low PON1 activity is an independent risk factor for atherosclerosis. Although

this finding is not universal (Eren et al., 2013) however low PON1 level is generally

reported in individuals who develop atherosclerosis (Mackness et al., 2006; Mackness and

Mackness 2012). PON1 plays multiple functions in prevention of atherosclerosis including

prevention of foam cells formation (Borowczyk et al., 2012; Farid and Horii, 2012;

Litvinov et al., 2012; Yilmaz 2012), reducing oxidative stress in lipoproteins (Gordon et

al., 2011) and inhibition of cholesterol synthesis in macrophages (de Goma et al., 2008).

Oxidation of LDL plays a major role in development of atherosclerosis (Berliner, 1996).

PON1 protects both LDL and HDL from oxidation by rendering antioxidant properties in

HDL, hydrolyzing lipid peroxides and preventing lipoperoxides accumulation in LDL thus

preventing atherosclerosis (Durrington et al., 2001; Mackness et al., 2002; Rosenblat et

al., 2006). PON1 may act directly on lipid peroxides or more probably lipid peroxides are

accumulated on HDL and then hydrolyzed by HDL associated PON1 (Aviram et al., 1998).

The inhibition of both LDL and HDL oxidation may contribute to protection against

cardiovascular disease (CVD). It has been postulated that paraoxonase (PON1) genotype

cvii

status may reflect anti-oxidant and antiatherogenic capacity of the enzyme (Mackness et

al., 1993; Rosenblat et al., 2006). In addition PON1 arylesterase/paraoxonase activities

are inversely related to the risk of coronary heart diseases and hypercholesterolemia

(Blatter et al., 1993; Humbert et al., 1993; Garin et al., 1997). A direct evidence of anti-

atherosclerosis function of PON1 comes from studies in animal models. Studies have

shown that PON1 deficient mice are more prone to atherosclerosis as compared to wild

type mice when both groups were fed with high fat and high cholesterol diets (Shih et al.,

1998). The current knowledge also indicates that PON1 is one of the major atheroprotective

agents (Mackness and Mackness, 2013). Role of PON1 needs to be explored further in

clinical studies for complete understanding of its regulation in humans.

Low density lipoprotein receptor (LDLR) is a receptor protein located on the

surface of plasma membranes of nucleated cells, especially in liver. This is a receptor

protein for LDL-C. Nobel Prize winners Goldstein and Brown have demonstrated that

LDLR plays vital role in cholesterol regulation and processes involved in atherosclerosis

(Goldstein and Brown, 2009). LDLR gene mutation is one of the major contributors

responsible for hypercholesterolemia while hypercholesterolaemia itself is also a cause of

atherosclerosis (Al-Allaf et al., 2010; Seijkens et al., 2014). Genome-wide association

studies have also indicated that LDLR gene mutations are strongly associated with

atherosclerosis (Katherisan et al., 2009). It is reported that quercetin consumption up-

regulates PON1 expression leading to increased protection against LDL oxidation (Gong

et al., 2009). Recently it was reported that increased level of serum PON1 activity in LDLR

(-/-) mice (due to consumption of quercetin and moderate ethanol) significantly inhibits

progression of atherosclerosis (Leckey et al., 2010). However, role of PON1 activity and

genotypes in LDLR mutated humans has not been studied previously. It is also indicated

previously (Ikonin, 2006) that genetic cause of 50-70 % of familial hypercholesterolaemia

remains unknown which indicates involvement of several unidentified genes in this

disease. Thus as an extension towards understanding the role of PON1 in familial

hypercholesterolaemia and protection against atherosclerosis, we report here the PON1

coding sequence SNPs (PON1 L55M and PON1 Q192R) and resultant paraoxonase and

arylesterase activity in hypercholesterolemia patients with mutated LDLR gene in a

Pakistani population.

cviii

4.2. Patients and Methods

4.2.1 Subjects

First family comprising of LDLR mutated Familial hypercholesterolemia patients

from Pakistani population, reported by our research group (Ajmal et al., 2010), was

included in this study. The family (n = 34) consisted of 10 LDLR mutated patients suffering

from hypercholesterolemia and 24 normal healthy individuals. An insertion mutation (c.

2416_2417InsG) was identified and reported previously (Ajmal et al., 2010) in exon 17 of

the LDLR gene in all of these patients. The study was approved by the Ethics Committee

and Institutional Review Board of the Department of Biosciences, COMSATS Institute of

Information Technology, Islamabad. All patients and controls were informed about the

study in their local language and written consent was also obtained from them prior to

inclusion in the study.

4.2.2. Methods

Methods were same as described for case-1. Results for all continuous variables are

expressed as Mean ± SD. Differences among patients and normal controls were assessed

by using χ2 test and Student’s t-test. P values <0.05 were considered as statistically

significant.

cix

4.3. Results

4.3.1. Demographic characteristics

Demographic characteristics with lipid profiles are summarized in Table 4.1. No

significant differences were present between patients and controls with respect to Mean ±

SD values of age, gender and body mass index (BMI). Routine laboratory findings of lipid

profile indicated significant differences between the patients and controls for TC (P <

0.0001) and LDL-C (P < 0.0001) while differences between the TG (P = 0.28) and HDL-

C (P = 0.084) were not significant. Paraoxonase (P = 0.001) and arylesterase activities (P

= 0.002) were significantly lower in patients compared to controls.

Table 4.1: Blood chemistry and clinical data of the affected and normal individuals of the studied

family. (Lipid profiles are previously reported by Ajmal et al., 2010)

Characteristics Patients

(n = 10)

Control

(n = 24)

p- value

Age (Years)

Male (Male: Female)

BMI (Kg/m2)

TC (mg/dl)

TG (mg/dl)

LDL-C (mg/dl)

HDL-C (mg/dl)

22.5±16.8

5:5

20±3.2

422.2±181.5

167.6±67.1

323.6±149.6

38±8.4

26.5±14.6

12:12

21±3.6

184.9±32.5

135.7±80.1

108.5±26.8

45.0±11.0

NS

NS

NS

< 0.0001

0.28

< 0.0001

0.084

Values are given in Mean ± S.D

cx

4.3.2. Pedigree and PCR-RFLP results

Pedigree is shown in (Fig. 4.1) with respective genotypes of the patients (filled

boxes and circles representing male and female patients, respectively) and healthy controls

(empty boxes and circles representing male and female controls, respectively). Pedigree

shows that the disease started from a brother III 3 and his sister III 2, both of which were

affected individuals. Three cases of consanguineous marriages were observed in this

family: (1): marriage between III 1 (healthy male with QQ/MM genotype) and III 2

(affected female with QQ/MM genotype), This marriage resulted in one affected male IV

4 with QQ/MM genotype and three healthy off springs. (2): marriage between III 3 male

patient and III 4 healthy female (QQ/LM genotype) resulted in 5 affected offsprings and 3

healthy offsprings. (3): marriage between two patients IV 4 (male) and IV 5 (female) (both

had QQ/MM genotype) resulted in three affected individuals (V4, V5, V6 with QQ/MM

genotype) and a healthy offspring V3 (QQ/MM genotype). Pedigree indicates

predominance of QQ/MM genotype in patients.

cxi

Fig. 4.1: Pedigree of hypercholesterolemia family (Filled boxes and circles represent male

and female patients respectively; Empty boxes and circles represent male and female

normal controls respectively). (Modified from Ajmal et al., 2010). The origin of LDLR

mutation associated hypercholesterolaemia is shown with red box. Consanguineous

marriage between a patient and normal individual is shown by blue box. Consanguineous

marriage between two patients is shown by green box. Respective PON1 coding region

polymorphism results of each individual are also shown.

cxii

4.3.3. Prevalence of PON1 genotypes

The prevalence of various genotypes (PON1 SNPs and combinations) is given in

Table 4.2 and 4.3 and comparison of genotypes is given in Table 4.4. PON1 L55 and R192

alleles were deficient in patients as compared to the healthy controls. Differences in PON1

L55M were less prominent as compared to differences in PON1 Q192R. QQ genotype was

highly prevalent in patients (90%) as compared to controls (41.7%). QQ/MM was

predominant in patients (80%) as compared to controls (33.3 %). QR/MM was more

prevalent in healthy individuals (54.2%) as compared to patients (0%). Graphical

presentation of combined genotypes of patients and healthy controls of the family

compared with healthy Punjabi population revealed a limited genotype pool in the cases as

well as controls as compared to healthy Punjabi population. Five of nine combined

genotypes (RR/LL, RR/LM, QQ/LL, QR/LM, QQ/LL) were absent in healthy controls of the

family while six of nine combined genotypes (RR/LL, RR/LM, QR/LL, QR/LM, RR/MM

and QQ/LL) were absent in patients. All the nine possible genotypes were present in healthy

Punjabis (Fig 4.2).

cxiii


in healthy controls of Punjabi hypercholesterolaemia family (n=24).

Polymorphism Allele

n = 48

Frequency Genotype

n = 24

Frequency

n % n %

PON1 Q192R Q

R

33

15

68.8

31.2

QQ

QR

RR

10

13

1

41.7

54.2

4.2

PON1 L55M L

M

2

46

4.16

95.83

LL

LM

MM

0

2

22

0

8.3

91.7


in hypercholesterolaemia patients of Punjabi family (n=10).

Polymorphism Allele

n = 20

Frequency Genotype

n = 10

Frequency

n % n %

PON1 Q192R Q

R

18

2

90

10

QQ

QR

RR

9

1``

0

90

10

0

cxiv

PON1 L55M L

M

2

18

10

90

LL

LM

MM

0

2

8

0

20

80

Table 4.4: Paraoxonase-1 allele and genotype distributions of L55M and Q192R

polymorphisms in patients and normal controls of hypercholesterolaemia family.

Characteristics

(Genotypes)

Patients

(n = 10)

Controls

(n = 24)

Q

R

L

M

LL

LM

MM

QQ

QR

RR

QQ/MM

18 (90%)

2 (10%)

2 (10%)

18 (90%)

0 (0%)

2 (20%)

8 (80%)

9 (90%)

1 (10%)

0 (0%)

8 (80%)

33 (68.75%) a

15 (31.25%) a

2 (4.16%)

46 (95.83%)

0 (0%)

2 (8.3%) a

22 (91.7%)

10 (41.7%) a

13 (54.2%) a

1 (4.2%)

8 (33.3%) a

cxv

QQ/LM

QR/LM

QR/MM

RR/MM

1 (10%)

1 (10%)

0 (0%)

0 (0%)

2 (8.3%)

0 (0%)

13 (54.2%) a

1 (4.2%)

a: Significant difference (P<0.05) compared with controls

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

Punjabi healthy


cxvi

Fig. 4.2: Comparison of combined genotype frequencies between healthy Punjabi

population, normal healthy controls and hypercholesterolaemia patients


Serum arylesterase activities (U/mL) of patients were significantly (P<0.05) low in

patients of QQ, QR, LM and LL genotypes as compared to healthy controls of the family

as well as when compared with healthy Punjabi population. Genotypes RR and MM were

absent in patients.

LL LM MM RR Q

R QQ

0

10

20

30

40

50

60

70

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

LL LM MM RR Q

R QQ

0

10

20

30

40

50

60

70

80

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

Healthy members

of studied Punjabi

family (n = 24)

Hypercholesterolaemia

patients of studied

Punjabi family (n = 10)

cxvii


of family (n = 24) and healthy Punjabi population (n = 140) with respect of QQ, QR, RR,

LL, LM, MM genotypes. a: statistically significant differences between patients and

normal controls of the family. b: statistically significant differences between patients and

healthy Punjabi population.


Serum paraoxonase activity in patients was significantly low in patients when

compared with normal controls of the family as well as when compared with healthy

0

20

40

60

80

100

120

140

160

180

200

QQ QR RR MM LM LL

PON1 genotypes

Seru

m A

ryle

ste

rase a

cti

vit

y

(U/m

L)

Controls Patients Healthy Punjabis

a, b

a, b

a, b

a, b

cxviii

Punjabi population for genotypes QQ, QR, LM and LL. Genotypes RR and MM were

absent in patients.

Fig. 4.4: Comparison of paraoxonase activity (U/L) between cases (n = 10) and healthy

controls of family (n = 24) and healthy Punjabi population (n = 140) with respect of QQ,

QR, RR, LL, LM, MM genotypes. a: statistically significant differences between patients

and normal controls of the family. b: statistically significant differences between patients

and healthy Punjabi population.

0

100

200

300

400

500

600

QQ QR RR MM LM LL

PON1 genotypes

Seru

m P

ara

oxo

nase a

cti

vit

y

(U/L

)

Series1 Series2 Series3

a, b

a, b

a, b

a, b

cxix


Only three combined genotypes (QQ/LM, QQ/MM, QR/LM) were present in

patients. Serum arylesterase activities (U/mL) were significantly low in patients as

compared to healthy controls of the family for QQ/LM and QQ/MM genotypes. QR/LM

genotype was absent in healthy controls of the family however Serum arylesterase

activities (U/mL) were significantly low in patients of QR/LM genotype as compared to

healthy Punjabis.



genotypes. a: statistically significant differences between patients and normal controls of

the family. b: statistically significant differences between patients and healthy Punjabi

population.

0

50

100

150

200

250

300

QQ

/LL

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM


Seru

m a

ryle

ste

rase a

cti

vit

y

Control Patients Healthy Punjabis

a, b

a

b

cxx


Serum paraoxonase activities (U/L) were significantly low in patients as compared

to healthy controls of the family in case of QQ/LM and QQ/MM genotypes. QR/LM was

absent in healthy controls while serum paraoxonase activity of patients in this genotype

were significantly low as compared to healthy Punjabis.

Fig. 4.6 Comparison of Paraoxonase activity between cases (n = 10) and healthy controls


genotypes. a: statistically significant differences between patients and normal controls of

the family. b: statistically significant differences between patients and healthy Punjabi

population.

-50

50

150

250

350

450

550

QQ

/LL

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM


Seru

m P

ara

oxo

nase a

cti

vit

ies

Controls Patients Healthy Punjabis

a

a

b

cxxi

4.4. Discussions

Association of PON1 with familial hypercholesterolaemia has not been fully

understood although some of the previous studies have shown a relationship of PON1 with

simvastatin activity (Tormas et al., 2000) carotid arterial wall thickness (Leus et al., 2000),

HDL levels and Linoleic acid oxidation (van Himbergen et al., 2005) and probucol function

(Inagaki et al., 2012) in familial hypercholesterolaemia patients. This is the first study of

PON1 activity and coding region polymorphisms in a hypercholesterolemia family with

LDLR mutation while effect of LDLR mutation on PON1 activity is not reported

previously. PON1 genotype and activity is not being monitored in routine clinical practice

for management of hypercholesterolaemia. Understanding any protective role of PON1

activity in patients susceptible to atherosclerosis will help to take early preventive measures

for hypercholesterolaemic patients.

The contribution of PON1 in Cardiovascular Disease (CVD) is weak in healthy

population but it is known that genotypes with minor effects in general population may

have more pronounced effects in patients, for example in Familial Hypercholesterolemia

(FH) cases (Leus et al., 2001; Wiegman et al., 2004). Contribution of PON1 in the

inhibition of atherosclerosis might be more pronounced in FH patients because they are

more prone to develop atherosclerosis than the general population (Thomas et al., 2005).

Normal sequence of LDLR gene and intact structure of LDLR receptor protein (Fig

4.7) are important determinants of cholesterol levels. All the patients in the current study

carried LDLR mutation (insertion mutation (c. 2416_2417InsG)) which leads to a frame

shift and formation of a “premature stop codon at position 816 in exon 17”, due to which

truncated LDLR protein is produced. This truncated LDLR protein lacks 45 amino acid

residues of cytoplasmic tail of normal protein. Thus a defective receptor is produced which

lacks normal cytoplasmic tail segment of LDLR protein (Hobbs et al., 1990). As a result,

cxxii

feedback inhibition of cholesterol biosynthesis is compromised which leads to

accumulation of cholesterol in blood and elevation of TC and LDL-C levels (Stanley,

2008). Thus all the patients in currant study were hypercholesterolemic due to defective

LDLR protein as a result of LDLR mutation (Ajmal et al., 2010).

Cytoplasmic tail of

normal LDLR protein

Insertion mutation (c.2416_2417InsG) in

exon 17 results in loss of 45 amino acid

residues of normal cytoplasmic tail due to

formation of premature stop codon

Site of insertion

mutation

(c.2416_2417InsG)

in exon 17

cxxiii

Fig. 4.7: Schematic diagram showing normal LDLR gene (A), mRNA (B), LDLR protein

(C) and respective portions of protein encoded by exons of the gene. (Al-Allaf et al., 2010).

Cytoplasmic tail of normal LDLR protein (shown in red box in this figure) is lost due to

premature stop codon formation as a result of insertion mutation (c. 2416_2417InsG) in

exon 17 of the patients included in our study.

Low PON1 activity has been reported in previous studies as one of the leading

factors causing atherosclerosis and myocardial infarction (Ayub et al., 1999; Mackness et

al., 2002; Van Himbergen et al., 2005). An association of low levels of PON1 activity with

atherosclerosis and cardiovascular diseases has been reported in previous studies (Jarvik et

al., 2000; Gur et al., 2006; Fatma et al., 2008). Therefore significantly decreased

paraoxonase (P = 0.001) and arylesterase (P = 0.002) activities in patients as compared to

the controls in present study indicate an increased risk of atherosclerosis in the patients.

Oxidation of LDL is one of the major contributors in atherosclerosis development

(Berliner, 1996). Dysfunctional LDLR protein of the patients (leading to cholesterol

accumulation) coupled with low serum paraoxonase and arylesterase activities (leading to

low protection of LDL against oxidative stress) in this study may have a synergistic effect

paving a way to development of atherosclerosis. One of possible evidence of this dual risk

scenario was observed in a patient (IV-4) in the studied family who had a history of CVD

in addition to FH. This patient had premature coronary artery disease and has suffered from

a MI at an early age which may have been due to decreased paraoxonase and arylesterase

activity because lipid profile of this 37 year old male was not too much elevated as

compared to other patients in this family.

Patient V-6 was identified with Xanthomas in addition to FH, but without any

history of CVD. However his levels of total cholesterol (TC = 917 mg/dl), low density

lipoprotein-cholesterol (LDL-C = 728 mg/dl) were markedly high and high density

lipoprotein-cholesterol (HDL-C = 22 mg/dl) was lower as compared to other FH patients

in the family. The presence of tendon xanthomas is a marker of high risk of CVD among

patients with FH, which along with decreased paraoxonase and arylesterase activities may

cxxiv

lead to atherosclerosis (Jarvik et al., 2000; Mackness et al., 2003; Gur et al., 2006;

Rosenblat et al., 2006; Fatma et al., 2008; Neslihan et al., 2008).

Low levels of serum paraoxonase and arylesterase activities of patients in this study

may be due to their genetic make up (PON1 coding region SNPs). The L55M

polymorphism affects the enzyme concentration (plasma PON1 protein levels indicated by

serum arylesterase activity), whereas the Q192R polymorphism determines the catalytic

efficiency (serum paraoxonase activity), but not the concentration (Bryk et al., 2005). The

PON1 M55 is associated with low level of plasma PON1 protein concentrations (Mackness

et al., 1998). The PON1 R192 allozyme is more rapid than Q192 in hydrolyzing paraoxon

(Costa et al., 2005). In our study the high prevalence of M55 allele in the patients may be

responsible for low levels of PON1 activity (Aviram et al., 2000). Frequency of the low-

activity allele (Q192) and unstable form (M55 isoform which is sensitive to proteolysis)

was collectively higher in patients of this family indicated by high prevalence of MMQQ

genotype (8, 80%) which may significantly reduce the function of PON1 for protection

against atherosclerosis. It is important to note that our study on healthy Pakistani Punjabi

male population (Case 1) revealed a very low frequency of MMQQ (4%) which indicates

that high prevalence of MM/QQ genotype (80%) in patients is not a sign of healthy status.

Indeed, PON1 phenotype (paraoxonase and arylesterase activities) has been

considered to be a better predictor of vascular disease than genotypes of PON1 due to

possible effects of other genetic and non-genetic factors (Jarvik et al., 2000; Rosenblat et

al., 2011). Upregulation of plasma PON1 levels by non-genetic factors (Rosenblat et al.,

2011) can have a potential advantage in such cases for better and achievable protection

against atherosclerosis. Thus our results combined with literature indicate the need of

regular monitoring and upregulation of serum paraoxonase activity in patients with

hypercholesterolemia for prevention of atherosclerosis. Our results add into the importance

of exploring PON1 as a therapeutic agent in order to accommodate the lower level of

plasma paraoxonase 1 in patients susceptible to atherosclerosis.

It was noted in the current study that LDLR mutated patients have low level of

PON1 which may possibly indicate some gene-regulation relations of LDLR with PON1

which needs to be investigated further.

cxxv

4.5. Conclusions

Both the hypercholesterolemia and PON1 deficiency are independent risk factors

for the development of atherosclerosis. In addition to controlling high levels of cholesterol,

there is a need to regularly monitor PON1 status of hypercholesterolaemic patients and

normal family members. Better understanding of factors up regulating PON1 status in

humans will have a significant public health impact by saving lives of patients who are

otherwise susceptible to atherosclerosis due to their deficient PON1 status. Our study

identified first ever relationship between LDLR mutation and PON1 genotypes and activity.

LDLR mutation was significantly associated with PON1 MM/QQ genotype and decreased

serum paraoxonase and arylesterase activities in patients as compared to controls.

Decreased serum PON1 activity may add the risk of atherosclerosis in patients who are

simultaneously hypercholesterolaemic due to defective LDLR protein. However as the

number of samples in the current study was small, studies on larger sample sizes are

expected to further elaborate relationship of PON1 with LDLR. But still the results are

important because this family is the sixth report worldwide of the LDLR mutation studied

in this family.

cxxvi

5.0. (Case study-4): Serum Paraoxonase and Arylesterase Activity in

Primary Glaucoma Patients

5.1. Introduction

Over 70 million people worldwide are affected by glaucoma (Thomas et al., 2007).

Most of the glaucoma patients belong to Asian populations (Morrison and Pollack, 2003).

Like many other countries (Quigley et al., 2006; Thomas et al., 2007), glaucoma is the

second leading cause of blindness (up to 23% of blindness cases) in Pakistani population

as well (Syed and Mughal, 1998).

Glaucoma was initially defined by Haik in 1959 as a disease of eye in which

Intraocular Pressure (IOP) is raised to such a level which is not tolerated by eye which

ultimately damages the eye (Rojanapongpun, 1999). Currently glaucoma is known as a

multifactor eye disease which is associated with optic neuropathy (Oliver and Cassidy,

2005), cupping of the optic disc, retinal ganglional cells’ degeneration (Neufeld, 1999),

visual field loss and decrease in color sensitivity (Mozaffarieh et al., 2008). These

degenerative changes associated with glaucoma are not reversible and result in thinning of

retinal nerve fiber layer (Mozaffarieh et al., 2008). If glaucoma is diagnosed at early stages

then it can be managed with medication, surgery and laser therapy (Wissinger et al., 2003).

However, glaucoma is called a silent blindness because majority of cases are not diagnosed

at early stages before occurrence of extensive damage (Anne, 1999).

Glaucoma is divided into two categories on the bases of IOP: normal tension

glaucoma (IOP<21 mmHg associated with optic neuropathy) and high tension glaucoma

(IOP>21 mmHg without optic neuropathy (Oliver and Cassidy 2005). Primary glaucoma

and secondary glaucoma are two categories based on pre-existing causative factors of

glaucoma. Primary glaucoma occurs due to unknown causes (mostly genetic factors) while

secondary glaucoma results from injury or other eye problems (Arthur and Jan, 2005). Two

cxxvii

major types of primary glaucoma include Primary Closed Angle Glaucoma (PCAG) and

Primary Open Angle Glaucoma (POAG) on the bases of open or closed anterior or drainage

angle (Quigley et al., 1996; Quigley et al., 2006). POAG is the most common type (90%)

of glaucoma cases (Oliver and Cassidy 2005). No noticeable symptoms appear in POAG

cases at early stages and gradually led to loss of vision (Anne, 1999). POAG occurs due to

blockage of aqueous outflow in trabecular meshwork resulting in increased IOP which in

turn damages the optic nerve (Adatia and Damji, 2005). POAG in one eye increases the

risk of its occurrence in the other eye (Distelhorst and Hughes, 2003). On the other hand

PCAG is a bilateral eye disease in which permanent loss of vision may occur within hours

due to rapid rise of IOP associated with partial or complete blockage of drainage through

trabecular meshwork (Kang et al., 2003a, b).

Prevalence of glaucoma is increased in cases of diabetes mellitus and myopia in

addition to family history of glaucoma (Morrison and Pollack, 2003). Family history of

glaucoma is a very strong risk factor increasing the chance of disease 4 times as compared

to individuals with no family history of glaucoma which is suggestive of genetic factors in

this disease (Taylor and Keeffe, 2001; Adatia and Damji, 2005). At least 4 genes (MYOC,

CYP1B1, WDR36 and OPTN) and 23 loci are reported to be associated with glaucoma

(Kumar et al., 2007; Yen et al., 2008). However, molecular mechanism of glaucoma is still

not clearly understood because of the lack of knowledge about cellular and biochemical

processes involved in regulation of IOP and retinal ganglional cell functions (Wiggs,

2007). Inagaki et al., (2006) studied PON1 gene polymorphisms in Japanese patients with

POAG and NTG. Results showed significant association of PON1 L55M with old age in

NTG patients suggesting that PON1 gene polymorphisms may influence clinical features

of glaucoma in Japanese patients. However, Inagaki et al., (2006) did not study PON1

phenotypes which may have revealed any additional information about presence or absence

of association of PON1 phenotype with glaucoma. No such studies are reported in any

other country to the best of our knowledge. Thus, we studied PON1 gene polymorphism

as well as serum paraoxonase and serum arylesterase activities in Pakistani glaucoma

patients (POAG and PCAG) in order to explore any role of PON1 gene in Pakistani

population.

cxxviii

5.1.1. Normal eye structure and flow of Aqueous humor

Aqueous humor is a clear watery fluid in the eye which maintains the IOP and

provides glucose, oxygen and metabolites including amino acids to lens and cornea

(Agarwal et al., 2003). Cellular debris, carbon dioxide, lactic acid and other waste products

are removed by aqueous humor. Formation and drainage of aqueous humor in eye is very

essential for the maintenance of normal IOP and normal function of lens and cornea

(Morrison and Pollack, 2003).

cxxix


normal IOP (Intra Ocular Pressure) for normal eye function.

(http://www.kayurshaheyemd.com/glaucoma-1)

5.1.2. Difference between normal eye and glaucoma

Normal function of drainage channel leads to normal flow of aqueous humor which

maintains IOP within normal physiological range (<21 mm Hg). Blocked drainage channel

leads to inhibition of fluid drainage which results in increased IOP due to retained fluid

(Oliver and Cassidy, 2005). Glaucoma is suspected at IOP value of >26 mm Hg while IOP

values >32 mm Hg leads to clear diagnosis of glaucoma (Anne, 1999). IOP values of 20-

30 mm Hg leads to slow and progressive damage over several years while 40-70 mm Hg

IOP values lead to rapid loss of vision (Karen, 2006).


normal IOP (Intra Ocular Pressure) for normal eye function

(http://www.webeca.com/content/vision%20problems/Glaucoma.htm)

http://www.kayurshaheyemd.com/glaucoma-1

http://www.webeca.com/content/vision%20problems/Glaucoma.htm

cxxx


normal IOP (Intra Ocular Pressure) for normal eye function.

(http://www.webeca.com/content/vision%20problems/Glaucoma.htm)

5.1.3. Difference between OAG and CAG

Open angle glaucoma is associated with normal anterior chamber angle of eye

(angle between cornea and iris) (Morrison and Pollack, 2003). POAG occurs due to

blockage of aqueous flow deep within trabecular meshwork. This blockage results in raised

IOP leading to optic nerve damage (Adatia and Damji, 2005). PCAG is characterized by a

narrow or closed angle between iris and cornea which occurs due to pupil dilation and

occlusion of trabecular meshwork (Morrison and Pollack, 2003). It causes resistance and

partial or complete blockage of drainage of aqueous humor through trabecular meshwork

and it may lead to permanent loss of vision within hours due to rapid rise in IOP (Kang et

al., 2003).

http://www.webeca.com/content/vision%20problems/Glaucoma.htm

cxxxi


normal IOP (Intra Ocular Pressure) for normal eye function

(http://www.drugs.com/health-guide/glaucoma.html)

5.1.4. Optic nerve damage due to glaucoma

Loss of optic nerve tissue due to axon degeneration and collapsed plates of lamina

ceribrosa leads to cupping of optic nerve head (ONH) and thinning of disc rim (Adatia and

Damji, 2005). As a result cup to disc ratio (c/d) is increased and nerve fiber layer becomes

defective (Oliver and Cassidy, 2005). Constriction of the blood vessels (vasospasm) due to

IOP leads to decreased blood supply to ONH and retinal ganglions which results in ONH

damage in glaucoma (Harris et al., 2008).

http://www.drugs.com/health-guide/glaucoma.html

cxxxii

Normal optic disc Moderate glaucoma Advanced glaucoma

Vertical c/d ratio: 0.2 Vertical c/d ratio: 0.7 Vertical c/d ratio: 0.99

Fig. 5.5: Diagram showing damage to optic nerve with advancement of glaucoma (Buorne,

2012)

(http://lceyes.com/wp-content/uploads/2013/07/Effects-of-Glaucoma.png)

5.1.5. Altered visual field due to glaucoma

Optic nerve defects due to glaucoma lead to abnormal visual field in glaucoma

patients. Vision is lost in those specific parts of Visual field which relate with anatomic

position of damaged/lost nerve fiber axons in eye. It results in tunneling of vision (loss of

http://lceyes.com/wp-content/uploads/2013/07/Effects-of-Glaucoma.png

cxxxiii

peripheral vision) (Arthur and Jan, 2005; Karen, 2006). Visual filed decreases with

advancement of glaucoma with the passage of time.

Fig. 5.6: Diagram showing Tunneling vision (loss of peripheral vision) with advancement

of glaucoma

(http://www.parekheyehospital.com/patientEducation/Glaucoma_2.jpg)

5.2. Patients and Methods

http://www.parekheyehospital.com/patientEducation/Glaucoma_2.jpg

cxxxiv

5.2.1. Subjects:

Pakistani Punjabi patients with POAG (n = 90), PCAG (n = 60) and healthy controls

(70) were included in this study. A complete ophthalmic examination of all patients was

done for diagnoses of glaucoma. Subjects with any other ocular disease were excluded.

Hypertensive, diabetic and cardiovascular disease patients were excluded. Control subjects

were non-hypertensive, non- diabetic without any cardiovascular disease. Clinical criteria

for diagnosis of POAG and PCAG are given in Table 5.1. Age and gender of participants

is shown in table 5.2

Table 5.1: Clinical criteria of screening of study participants for glaucoma (Michael et al.,

2008)

Opthalmic

examination

POAG PCAG Normal controls

1-Slit lamp

biomicroscopy

2- Visual acuity

(with the help of

Snell’s chart)

3- Visual field

defects (determined

by Humphrey 30-2)

4- IOP (by

Goldman

Applanation

Tonometer)

1- IOP: >21 mmHg

2- Diffuse or focal

thinning of disc

rims and >0.5 cup

to disc ratio

3- Visual field

defects of glaucoma

4- Open anterior

chamber angle

1- IOP: >21 mmHg

2- Diffuse or focal

thinning of disc

rims and >0.5 cup

to disc ratio

3- Visual field

defects of glaucoma

4- Closed anterior

chamber angle

1-IOP: <21 mm Hg

2- c/d ratio: < 0.5

3- No visual field

defects

4- Normal anterior

chamber angle

5- No history of any

eye disorder

cxxxv

5- cup to disc ratio

(c/d) (by indirect

fundoscopy)

Cup to disc ratio (c/d) was determined by dividing diameter of cup (inner portion

of optic nerve head) by diameter of disc (outer portion of optic nerve head) as illustrated

in the Fig 5.7.

Fig. 5.7: Determination of c/d (cup/disc ratio) of optic nerve head (ONH) (Normal ONH

is shown in this figure) (Buorne, 2012)

cxxxvi

Table 5.2: Demographic characteristics of study participants (Michael et al., 2008)

Characteristics POAG (n = 90) PCAG (n = 60) Normal controls

(n = 70)

Age: (Mean)

Male

Female

57.9±14.3

64.4%

35.5%

57±10.05

51.67%

48.3%

50.9±8.1

66%

34%

Results of PON1 genotypes and phenotypes were compared between age and

gender matched normal controls and POAG and PCAG patients. In addition, results of

these three groups were compared with the results of healthy male Punjabi population (n =

140, male = 100%; female = 0%; mean age = 30± 10 years)

5.2.2. Methods:

Methods were same as described for case-1 (Chapter 2.0; pages 16-19).

cxxxvii

5.3. Results:

Results of PON1 coding region polymorphism (Table 5.3-5.5) showed

predominance of PON1 192Q in healthy controls (61%), POAG patients (63.5%) and

PCAG patients (66%) in case of PON1 Q192R polymorphism while PON1 55L was

predominant in healthy controls (64%), POAG patients (73.5%) and PCAG patients (68%)

in case of PON1 L55M polymorphism. PON1 QQ genotype was predominant (over QR

and RR genotypes) in normal controls (47%) POAG (50%) and PCAG cases (50%). PON1

LL genotype was predominant (over LR and RR genotypes) in normal controls (45%),

POAG (57%) and PCAG patients (49%).


in age and gender matched healthy controls (non-glaucomic) (n=70).

Polymorphism Allele

n = 140

Frequency Genotype

n = 70

Frequency

n % n %

PON1 Q192R Q

R

86

54

61.43

38.57

QQ

QR

RR

33

20

17

47.14

28.57

24.28

cxxxviii

PON1 L55M L

M

89

51

63.57

36.43

LL

LM

MM

31

27

12

44.28

38.57

17.14


in POAG patients (n=90).

Polymorphism Allele

n = 180

Frequency Genotype

n = 90

Frequency

n % n %

PON1 Q192R Q

R

114

66

63.33

36.66

QQ

QR

RR

45

24

21

50

26.66

23.33

PON1 L55M L

M

132

48

73.33

26.66

LL

LM

MM

51

30

9

56.66

33.33

10

cxxxix


in PCAG patients (n=60).

Polymorphism Allele

n = 120

Frequency Genotype

n = 60

Frequency

n % n %

PON1 Q192R Q

R

79

41

65.83

34.17

QQ

QR

RR

30

19

11

50

31.67

18.33

PON1 L55M L

M

81

39

67.5

32.5

LL

LM

MM

29

23

8

48.33

38.33

13.33

5.3.1. Comparison of PON1 genotypes distribution

Comparison of PONI coding region genotypes between patients of POAG and

PCAG with age and gender matched normal controls (Table 5.6) did not show any

significant differences between patients and controls. Similarly, genotypes patterns of

POAG, PCAG and controls did not show any significant differences from normal healthy

male Punjabi population. 3D graphic presentation revealed pattern of POAG, PCAG and

normal controls which was similar to the pattern of genotypes observed for healthy male

Punjabi population. Differences of PON1 genotypes across these four groups were non-

significant (Fig. 5.8).

cxl

Table 5.6: Comparison of PON1 genotypes between controls and glaucoma patients. a

Genotypes Punjabis

(n=140)

Controls (70) POAG (n=90) PCAG (n=60)

Q

R

L

M

QQ

QR

193 (68.93%)

87 (31.07%)

207 (73.93%)

73 (26.07%)

76 (54.29%)

43 (30.71%)

85 (61%)

55 (39%)

90 (64%)

50 (36%)

33 (47%)

20 (28%)

114 (63.5%)

66 (36.5%)

132 (73.5%)

48 (26.5%)

45 (50%)

24 (27%)

79 (66%)

41 (34%)

82 (68%)

38 (32%)

30 (50%)

19 (32%)

cxli

RR

LL

LM

MM

QQ/LL

QQ/LM

QQ/MM

QR/LL

QR/LM

QR/MM

RR/LL

RR/LM

RR/MM

21 (15%)

74 (52.86%)

59 (42.14%)

7 (5%)

34 (24.29%)

35 (25%)

5 (3.57%)

27 (19.29%)

14 (10%)

2 (1.43%)

12 (8.57%)

9 (6.43%)

2 (1.43%)

17 (25%)

31 (45%)

27 (38%)

12 (17%)

13 (19%)

15 (21%)

5 (7%)

10 (14%)

7 (10%)

3 (4%)

8 (12%)

5 (7%)

4 (6%)

21 (23%)

51 (57%)

30 (33%)

9 (10%)

23 (26%)

16 (18%)

5 (6%)

19 (21%)

5 (6%)

0 (0%)

9 (10%)

8 (9%)

4 (4%)

11 (18%)

29 (49%)

23 (38%)

8 (13%)

13 (22%)

15 (25%)

2 (3%)

11 (18%)

5 (8%)

4 (6%)

5 (9%)

3 (5%)

2 (4%)

a: Differences between patients and controls and between patients and healthy male

Punjabis were Statistically non significant

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ Punjabi healthy


cxlii

Fig. 5.8: Comparison of genotypes between Glaucoma patients (PCAG and POAG) with

normal controls and healthy male Punjabi population


Distribution of serum arylesterase activities (U/mL) according to PON1 L55M and

PON1 Q192R revealed no significant differences between glaucoma patients (POAG and

PCAG) and age and gender matched normal controls. However, values of serum

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

LL LM MM RR Q

R QQ

0

5

10

15

20

25

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

LL LM MM RR Q

R QQ

0

5

10

15

20

25

30

Prevalence (%)

PON1 L55M

PON1

Q192R

RR

QR

QQ

Normal controls

(n = 70)

PCAG patients

(n = 60)

POAG patients

(n = 90)

cxliii

paraoxonase activity of healthy male Punjabi population was significantly higher (P<0.05)

when compared with normal controls and patients of both types (POAG and PCAG) in

QQ, QR, RR, LM and LL genotypes (Fig. 5.9). Difference between normal controls and

healthy Punjabis was not significant in case of MM genotype.



genotypes. a: statistically significant difference (P<0.05) between healthy male Punjabis

and healthy controls. b: statistically significant difference (P< 0.05) between healthy male

Punjabis and POAG patients. c: statistically significant difference (P<0.05) between

healthy male Punjabis and PCAG patients. NS: Non significant differences between

controls and POAG or PCAG patients


The differences of serum paraoxonase activities (U/L) between glaucoma patients

(POAG and PCAG) and age and gender matched normal controls (of QQ, QR, RR, MM,

0

20

40

60

80

100

120

140

160

180

200

QQ QR RR MM LM LL

PON1 genotypes

Seru

m A

ryle

ste

rase a

cti

vit

y

(U/m

L)

Controls POAG PCAG Healthy Punjabis

a, b, c,

NS

a, b, c,

NS

a, b, c,

NS

b, c

NS

a, b, c,

NS

a, b, c,

NS

cxliv

LM, and LL genotypes) were not significant. Serum paraoxonase activity of healthy male

Punjabis of all genotypes (QQ, QR, RR, MM, LM, LL) were significantly higher (P<0.05)

than normal controls and glaucoma patients (POAG and PCAG) (Fig 5.10).



genotypes. a: statistically significant difference (P<0.05) between healthy male Punjabis

and healthy controls. b: statistically significant difference (P< 0.05) between healthy male

Punjabis and POAG patients. c: statistically significant difference (P<0.05) between

healthy male Punjabis and PCAG patients. NS: Non significant differences between

controls and POAG or PCAG patients


-50

50

150

250

350

450

550

650

QQ QR RR MM LM LL

PON1 genotypes

Seru

m P

ara

oxo

nase a

cti

vit

y

(U/L

)

Series1 Series2 Series3 Series4

a, b, c,

NS

a, b, c,

NS

a, b, c,

NS

a, b, c,

NS

a, b, c,

NS a, b, c,

NS

cxlv

Distribution of serum arylesterase activities (U/mL) according to combined

genotypes of PON1 L55M and PON1 Q192R did not show any significant differences

between glaucoma patients (POAG and PCAG) and age and gender matched normal

controls. However, serum arylesterase activity of healthy male Punjabi population was

significantly higher (P<0.05) than normal controls and patients (POAG and PCAG) in all

combined genotypes except RR/MM (Fig 5.11).

Fig. 5.11: Comparison of serum arylesterase activities (U/mL) of Glaucoma patients

(PCAG and POAG) with normal controls and healthy male Punjabi population according

to combined genotypes. a: statistically significant difference (P<0.05) between healthy

male Punjabis and healthy controls. b: statistically significant difference (P<0.05) between

healthy male Punjabis and POAG patients. c: statistically significant difference (P<0.05)

between healthy male Punjabis and PCAG patients. NS: Non significant differences

between controls and POAG or PCAG patients

020406080

100120140160180200

QQ

/LL

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM


Seru

m a

ryle

ste

rase a

cti

vit

y

Control POAG PCAG Healthy Punjabis

NS

a, b,

c, NS

a, b,

c, NS a, b,

c, NS

a, b,

c, NS

a, b,

c, NS a, b,

c, NS b, c,

NS a, b,

c, NS

cxlvi


Differences of serum paraoxonase activities (U/L) between glaucoma patients

(POAG and PCAG) and age and gender matched normal controls were not significant in

any of the combined genotypes of PON1 L55M and PON1 Q192R However, serum

paraoxonase activity of healthy male Punjabi population was significantly higher (P<0.05)

than normal controls and patients (POAG and PCAG) in all combined genotypes (Fig

5.12).



combined genotypes. a: statistically significant difference (P<0.05) between healthy male

Punjabis and healthy controls. b: statistically significant difference (P<0.05) between

healthy male Punjabis and POAG patients. c: statistically significant difference (P<0.05)

between healthy male Punjabis and PCAG patients. NS: Non significant differences

between controls and POAG or PCAG patients

-50

50

150

250

350

450

550

650

QQ

/LL

QQ

/LM

QQ

/MM

QR

/LL

QR

/LM

QR

/MM

RR

/LL

RR

/LM

RR

/MM


Seru

m P

ara

oxo

nase a

cti

vit

ies

Control POAG PCAG Healthy Punjabis

a, b,

c, NS a, b,

c, NS a, b,

c,

NS

a, b,

c, NS a, b,

c, NS a, b,

c, NS

a, b,

c, NS

a, b,

c, NS a, b,

c, NS

cxlvii

5.4. Discussions:

Glaucoma is a highly prevalent neurodegenerative eye disorder which is associated

with multiple of genetic and non-genetic factors. However, still complete mechanism(s)

involved in development of glaucoma are not clear. Most of the types of glaucoma

(congenital glaucoma and Reiger syndrome, etc.) can be mapped down to specific genes

responsible for deformed anterior eye chamber (Cohen and Allingham, 2004). However,

POAG can not be described by a single Mendelian mode of inheritance; rather it occurs as

a result of interaction of multiple of genes (Weinreb and Khaw, 2004). There are only some

remote glimpses in previous studies which may possibly relate PON1 with glaucoma,

although direct evidence is lacking. Many studies suggest that altered ocular circulation is

involved in OAG (Flammer et al., 1999; Henry et al., 1999; Fuchsjager-Mayrl, 2004) while

it is known that risk of atherosclerosis (leading to impaired blood circulation) is increased

with decreased serum PON1 activity. Statins and other cholesterol reducing agents are

associated with reduction of risk of OAG (McGwin et al., 2004). It is also reported that

PON1 is up-regulated by statins in vitro at gene level and serum PON1 level and activity

is increased in patients treated with statins (Deakin et al., 2003). Thus it appears that PON1

is somehow related with glaucoma, especially POAG. PON1 protects LDL from oxidative

modification while fluorescent labeled and modified LDL are incorporated into trabecular

meshwork cells (Chang et al., 1991) and trabecular meshwork malfunction is related with

glaucoma. This indirect relationship of PON1 with trabecular meshwork cells may also

indicate some role of PON1 in glaucoma. Inagaki et al., (2006) studied PON1 coding

region polymorphism in Japanese OAG patients (NTG and POAG). Although 55L allele

carrier NTG patients were significantly young as compared to 55M and IOP was

significantly higher in glaucoma patients with 192R allele, however differences in

genotypes were not significant between controls and any of patients group. Inagaki et al.,

(2006) suggested that clinical features of OAG in Japanese patients may be influenced by

cxlviii

PON1 gene polymorphisms. In order to further understand relationship of PON1 with

glaucoma, we studied PON1 coding region polymorphism alongwith serum paraoxonase

and arylesterase activities in Pakistani POAG and PCAG patients.

Prevalence of PON1 L55M and PON1 Q192R genotypes and allele frequencies in

POAG and PCAG (Q>R, L>M, QQ>QR>RR, LL>LM>MM) were same as observed in

age and gender matched controls and healthy male Punjabis. Differences of genotypes

across these four groups were not significant. Combined genotypes of QQ/LL, QQ/LM and

QR/LM were predominant in all these four groups with only non-significant differences

across the groups. Thus, our results did not reveal any significant differences of PON1

genotypes between glaucoma patients (POAG and PCAG), age and gender matched

controls and healthy male Punjabis which indicate that PON1 genotypes are not related

with glaucoma in Pakistani population which is in accordance with results of Inagaki et al.,

(2006) in Japanese population. Serum paraoxonase and arylesterase activities followed the

same trend of relationship with genotypes (RR>QR>QQ, LL>LM>MM) in POAG and

PCAG patients as was seen in normal controls and healthy Punjabis. Although serum

paraoxonase and arylesterase activities in age and gender matched controls were slightly

higher than POAG and PCAG cases in most of PON1 genotypes in our study on glaucoma

patients, however these differences were also not statistically significant. These results

apparently indicate that PON1 genotype and level of PON1 in serum (systemic blood

circulation) is not significantly associated with primary glaucoma in Pakistani population.

However, significantly higher levels of serum paraoxonase and arylesterase activity

(P<0.05) in healthy male Punjabi population were detected as compared to POAG patients,

PCAG patients and normal controls of this study. Lower activity in age and gender matched

healthy controls, POAG and PCAG patients compared with healthy male Punjabis may be

related to the main difference of age because healthy Punjabis were younger (Mean age

30± 10) as compared to POAG patients (age =57.9±14.3 years), PCAG patients (age=

57±10.05 years ) and normal controls (age= 50.9±8.1 years).

Increased age is a common factor for increasing risk of POAG and PCAG while

age is also one of the major determining factors for PON1 activity (Costa et al., 2005). Age

cxlix

related differences of PON1 activities observed in various studies in animal models and

humans (Li et al., 1997; Moser et al., 1998; Cole et al., 2003) suggest that serum (and liver)

PON1 activities increase with age till adulthood after which it becomes stable. No

differences of serum and liver PON1 levels were found in rats with aging after adulthood

(Karanth et al., 2000). Initially it was suggested by some studies that PON1 levels do not

change in humans after adulthood (Mueller et al., 1983). However, recent investigations

has established that PON1 activity progressively decreases with aging in elderly humans

(Milochevitch and Khalil, 2001; Jarvik et al., 2002; Seres et al., 2004). Glaucoma can occur

in all age groups (as it occurs in children as well as in elderly) however risk of glaucoma

increases with increase in age after adulthood. About 2% of adults of 40 years age suffer

from glaucoma while occurrence rises to 4% after 70 years of age (Oliver and Cassidy,

2005). Incidence of POAG in 50-59 years age group is 0.7% which rises to 1.8% after 60

years (60-69 years age group) and still rises further to 3.2% after the age of 70 years (70-

79 years age group) (Adatia and Damji, 2005). Thus age is considered a potent risk factor

for glaucoma (Stephen, 2008) however molecular effect of age on the development of

glaucoma is not known. Our results indicate that age dependant decrease in serum

paraoxonase and arylesterase activities might explain molecular bases of age as a risk factor

for development of glaucoma.

In addition to difference of age, all the healthy Punjabis were male (100%) as

compared to other groups (normal controls, POAG and PCAG patients) which included

male (51.67% to 66%) as well as female (34% to 48.3%) participants. This difference of

gender distribution may suggest some influence in our results. Although serum PON1 level

is affected by gender in mice, however genetic heterogeneity in humans is considered to

obscure difference of PON1 levels between male and females (Mueller et al., 1983;

Wehner et al., 1987; Ali et al., 2003; Costa et al., 2005). Thus, gender differences between

POAG and PCAG patients and healthy male Punjabis might not affect serum paraoxonase

and arylesterase activities.

cl

5.5. Conclusions:

The results indicate that PON1 genotype is not involved in primary glaucoma in

Pakistani population. However, significant differences of serum paraoxonase and

arylesterase activities in glaucoma patients and healthy controls compared with younger

population suggests age dependant association of PON1 with glaucoma. Studies on local

(rather than systemic) PON1 protein levels and activities in eyes especially in trabecular

meshwork may reveal local effect of PON1 enzyme on eye tissues involved in glaucoma.

6.0 Detection of Methyl Parathion by Using Purified Green

Fluorescent Protein (GFPuv) as a Biosensor Molecule

6.1 Introduction

Methyl parathion is one of the highly toxic organophosphate pesticides. Its

extensive use inevitably contaminates the environment thus adversely affecting natural

ecosystems and producing toxic effects in non-targeted life (living organisms other than

targeted insect pests). Thus its detection in the environment is necessary for identification

and ultimate detoxification of the contaminated areas for prevention of exposure to humans

and other non-targeted species.

Methyl parathion detection needs sophisticated equipments and skilled manpower

and these methods are relatively costly which makes them inaccessible to remote

agricultural areas, especially in developing countries. These methods include Gas

Chromatography Mass Spectroscopy (GC-MS), (Goncalves et al., 2004) High-

cli

Performance Liquid Chromatography (HPLC), (Papadopoulou and Patsias, 1996)

electrochemical (Hart, et al., 1997), spectrophotometric (Naidu et al., 1990) and Fourier

transform (FT) Raman methods (Skoulika and C. A. Georgiou, 2000, Sato-Berru, et al.,

2004; M. Alak-Ala and T. Vo-Dinh, 1987) (Li et al., 2010). Some drawbacks of these

methods are indirect measurement, lengthy sample preparation, high cost, need of skilled

manpower, and non-portability. Thus an easy to use portable biosensor system requiring

lesser production and operative costs will be highly beneficial for on spot detection of

methyl parathion over large agricultural lands in the agricultural countries.

Fluorescent proteins are excellent tools which can be used in the field of biosensing.

Wild type and engineered fluorescent proteins have been used previously as biosensor

molecules for detection of copper and other metals (Todd et al., 2000; Yasmeen et al.,

2007; Yasmeen et al., 2008; Emese et al., 2013), bacterial endotoxins (Yan et al., 2002)

and superoxide radicals (Fadi et al., 2006) on the basis of fluorescence quenching assays.

Fluorescence quenching based biosensing assays provide a quantitative measure of the

analytes on the basis of quantification of decrease in fluorescence intensity. In this chapter,

we describe purification of GFPuv and its characterization for methyl parathion biosensing

application.


6.2.1. Selection of Fluorescent protein

We searched literature for identifying possible binding motifs of methyl parathion

and other OP compounds which may be added in fluorescent proteins for possible

application in GFP based biosensing application. Literature search revealed that many of

plasma proteins bind to OPs at tyrosine residues (Oksana and Lawrence 2010). Fluorescent

Proteins with tyrosine residues located within potential OP binding motifs may be ideal

candidates for fluorescence quenching based detection of methyl parathion. Several

tyrosine residues are already present on the surface of a well characterized and highly stable

green fluorescent protein (GFPuv). Thus we expressed and purified GFPuv protein for

exploring its utility as a biosensor molecule for detection of methyl parathion.

clii

6.2.2. Expression and purification of GFPuv

GFPuv plasmid (pGFPuv - Clontech) was transformed into E. coli DH5α cells. The

transformed cells were incubated overnight at 37 oC in agar plates containing ampicillin

(100 μg/mL). Glycerol stocks were made from a single isolated colony. GFPuv expression

was optimized for high yield of GFPuv protein. We used the method of Li et al., (2001)

with some modifications for Nickel affinity chromatography and combined it with FPLC

for GFPuv purification. Bacterial cells were grown overnight in 100 mL LB broth with

ampicillin (100 μg/mL) at 37 oC at 200 rpm. An aliquot of 50 mL resultant culture was

added in each 1 L LB broth supplemented with ampicillin. The culture was allowed to grow

at 37 oC in shaking incubator (180 rpm) until OD600 nm = 0.6. The culture was cooled down

to 16 oC and allowed to grow at 16 oC for 16 hours at 180 rpm. Cells expressing GFPuv

were collected by centrifuging at 6000 rpm (JLA 8.1000 fixed Angle Rotor, Beckman

Coulter) and resuspended in 50 mL of lyses buffer (1x PBS, pH= 7.5, 1M NaCl; Triton X

100, 5% (v/v) glycerol, 1 mM PMSF). Bacterial cells were lysed by sonication and lysed

cells were centrifuged at 18,000 rpm (JA-20 Fixed Angle Rotor, Beckman Coulter) for 30

minutes at 4 oC. The resultant supernatant was collected and subsequently mixed with 2

mL of Ni+2-NTA (Qiagen) resin (pre-equilibrated with lyses buffer) for binding at 4 oC.

After 1 hour incubation, the Ni+2-NTA resin was washed thrice with binding buffer (10

mL) containing 1X PBS (pH = 7.5) and 1M NaCl. Elution of GFPuv was carried out in

cold room (4 oC) under pH gradient with 10 mL elution buffer volumes at decreasing pH

values of 7.5, 7.0, 6.5 and 6.0. The eluted fractions were analyzed on a 12% SDS PAGE

gel. The eluted protein from Ni+2-NTA affinity chromatography was further purified

through the HiloadTM 16/60 SuperdexTM prep grade (Amersham Biosciences, Sweden) pre-

equilibrated in a gel filtration column with a buffer containing 1X PBS (pH 7.5), 200 mM

NaCl, 5% Glycerol. Chromatographic purification procedure was performed at 4 oC by

using the Amersham Biosciences AKTA FPLC.

6.2.3. Fluorescence assay

cliii

Modulus Luminometer/Fluorometer (Turner BioSystems, Synnyvale, CA) with

appropriate filters (ex: 395 nm, em: 509 nm) was used for fluorescence intensity

measurements. GFPuv protein was diluted in buffer (1X PBS, pH 7.5) to final

concentration of 100 nM and 50 nM. Samples containing GFPuv and methyl parathion

were incubated at room temperature for 30 minutes to allow stabilized interaction of methyl

parathion with GFPuv molecules. Effects of various concentrations of methyl parathion (0

– 200 μM) on fluorescence of GFPuv (100 nM and 50 nM) were noted and compared with

controls: (1) - GFPuv aliquots without addition of methyl parathion, and (2) - buffer

without GFPuv protein were used as controls. All the measurements were repeated in

triplicate.

6.3. Results:

6.3.1. Expression of GFPuv

Transformed E. coli DH5α colonies were screened for the presence of fluorescence

under UV. Results indicate that GFPuv is well expressed in E. coli DH5α cells as the

transformed cells were highly fluorescent when observed under UV light (Fig. 6.1).

cliv

Fig 6.1: Expression of GFPuv in E. coli DH5 α transformed with pGFPuv visualized under

UV light

6.3.2. IMAC purification of GFPuv

Fractions eluted from IMAC purification were run on 12% SDS PAGE gel. Results

showed that GFPuv was successfully eluted with higher purities at pH 7.0 as compared to

GFPuv

(26.867 kDa)

clv

other pH values (Fig. 6.2). Protein impurities were eluted at other pH values at a greater

extent as compared to pH 7.0.

Fig 6.2: IMAC: Nickel affinity chromatography without adding His tag in GFPuv protein.

1- Pellet (pH =7.5); 2- lysate (pH =7.5); 3- Elution (pH=7.5); 4- Elution (pH=7.0); 5-

Elution pH 7.0; 6- Protein marker; 7- Elution pH 6.5; 8-Elution pH 6.5; 9- Elution pH=6.0;

10- Elution pH=6.0

1 2 3 4 5 6 7 8 9 10

42.7 34.6 27.0 20.0 14.3 6.5

clvi

6.3.3 FPLC

Results of FPLC (Fig 6.3) showed a major peak in which all the aliquots were

fluorescent on exposure to UV. Aliquots from the central portion of the peak were selected

and checked for purity by running on 12% SDS PAGE gel. SDS PAGE gel revealed a

single band of purified native GFPuv protein (Fig 6.3). Finally eluted protein was

quantified and utilized for fluorescence quenching assay.

Peak with highly purified eluted GFPuv

Fluorescent protein with small

impurities

clvii

Fig 6.3: Purification of GFPuv. Fractions from the center of major peak were collected and

observed for presence of fluorescence. The eluted fractions were checked for purity with

help of running on 12% SDS PAGE. Highly purified GFPuv was obtained.

6.3.4 Fluorescence quenching assay

Results of fluorescence quenching assay (Fig 6.4) indicated a linear decrease in

relative fluorescens of GFPuv with respect to increasing concentration of methyl parathion.

Negative control (GFPuv without addition of methyl parathion) did not show any decrease

of fluorescence till the end of the experiment which indicated that fluorescence quenching

effect in test samples (GFPuv exposed to methyl parathion) was due to interaction of

GFPuv with methyl parathion. There was a small difference of regression values (y and

R2) in the simple linear regression analysis of the data for two different concentrations (100

nM and 50 nM) of biosensor molecule (GFPuv).

y = -0.0022x + 1.0336

R2 = 0.9721

0

0.2

0.4

0.6

0.8

1

1.2

0 100 200 300

Methyl parathion (μM)

Relative

Fluorescence

GFPuv without MP Buffer without GFPuv and MP GFPuv with MP

100 nM

GFPuv

RFU

clviii

Fig. 6.4: Fluorescence quenching effects of methyl parathion (0-200 μM) on purified two

different concentrations (100 nM and 50 nM) of GFPuv protein indicating a biosensing

effect. (RFU: Relative Fluorescence Units)

6.4. Discussions

Unique biophysical properties of green fluorescent protein of Aequorea victoria

provide opportunity for its use as a fluorescent marker in diverse areas of biotechnology

including biosensors. GFPuv is a mutant GFP extensively used in the biotechnology.

GFPuv is one of the most favored fluorescent proteins in biotechnology applications due

to its improved properties (45 times brighter than wild type GFP). In addition, GFPuv is

well expressed in E. coli DH5α under different growth conditions (Penna et al., 2004)

resulting in a high yield of protein.

GFPuv binds with Nickel beads due to its metal affinity due to its own Histidine

residues as described by Li et al., (2001). Li et al., (2001) previously reported purification

of GFPuv with the help of IMAC via Ni (II), Cu (II), Zn (II) and Co (II). We developed

our method of GFPuv purification on the basis of the results of Li et al., (2001) with some

modifications while we also included an additional step of FPLC in order to ensure high

purity of finally eluted protein. GFPuv was eluted from Nickel beads during purification

on the basis of washing the beads with buffers of decreasing pH (pH gradient). The results

indicate that GFPuv can be purified without adding Histidine tags.

y = -0.0026x + 0.9477

R2 = 0.98350

0.2

0.4

0.6

0.8

1

1.2

0 100 200 300

Methyl parathion (μM)

Relative

Fluorescence

GFPuv without MP Buffer without GFPuv and MP GFPuv with MP

50 nM

GFPuv

RFU

clix

Methyl parathion solubility in water is 50 mg/L (Falicia et al., 2005). Methyl

parathion half life in a model aquatic ecosystem was reported as 9-17 days (Mabury et al.,

1996). Its half life in water is 24-35 days and 9-11 days in soil (Sakellarides et al., 2002),

and hence methyl parathion occurs at low concentration in soil and water of contaminated

areas (0.01 to 2.5 μg/L in water samples and 128 mg/kg soil samples of contaminated

Pakistani regions) (Ahad et al., 2000; Tariq et al., 2004; Ahad et al., 2006; Nafees et al.,

2008). Detection of such a low level of pesticide from the environmental samples in the

field conditions is the real challenge. Our results indicate that GFPuv is able to detect low

levels of methyl parathion (0- 200 μM) on the basis of fluorescence quenching. There was

a linear decrease in fluorescence intensity with increasing concentration of methyl

parathion which indicates quantitative relationship between methyl parathion and

biosensor signal (fluorescence intensity).

Our results suggested fluorescence inhibition of GFPuv with methyl parathion. In

order to identify possible binding sites of methyl parathion with GFPuv, we searched the

literature for binding patterns of methyl parathion and/or other OPs with proteins. It is

known previously that methyl parathion and other OPs bind to several plasma proteins

(Silva et al., 2004). Primary binding site of methyl parathion is tryptophan residue 214 and

212 of human and bovine serum albumin respectively (Silva et al., 2004). Other studies on

OPs more conclusively identified binding motifs containing serine or tyrosines with

positively charged amino acids located in vicinity. The proteins of serine hydrolase family

react most rapidly with OPs (Richards et al., 2000; Casida and Quistad, 2004). The amino

acid residue labeled in these enzymes by OPs is the active site serine. These serine residues

occur in the consensus sequence of GXSXG. Another group of OPs binding proteins

revealed OP binding to tyrosine; for example papain and bromelain bind DFP on tyrosine

(Murachi et al., 1965; Chaiken and Smith, 1969). OP binding to tyrosines has also been

reported previously in albumin and tubulin (Li et al., 2007; Li et al., 2008). In addition to

GXSXG, RYTR and SYSM are two other known motifs which bind OPs (Lockridge and

Schopfer, 2010). The common feature in the motif for OP-reactive tyrosines (Table 6.1) is

the presence of a positively charged arginine or lysine within 5 residues of the tyrosine.

Tyrosine form a covalent bond with OP in both peptide motifs (RYTR and SYSM),

however the reaction proceeded more readily with RYTR than with SYSM (Li et al., 2009).

clx

Dichlorvos, chlorpyrifos and DFP are reported to bind a small synthetic peptide motif

RYTR at Y residue (Li et al., 2009; Lockridge and Schopfer, 2010). Thus Tyrosine residues

flanked by positively charged amino acids are the most possible sites for binding methyl

parathion and other OP’s. Based on the literature on OP binding motifs combined with our

fluorescence quenching results, we hypothesize that solvent exposed tyrosine residues of

GFPuv flanked by positively charged amino acids are the potential expected sites of

interaction with methyl parathion. Tyrosine residues in RYPDHMKRH, GNYKTR and

HKLEYNYNSHNV motifs of GFPuv are more likely to be interacting sites due to being

close to positively charged amino acids shown in yellow highlights (Fig 6.5). Such

potential candidate Tyrosine residues of GFPuv (Fig 6.5) exposed to the solvent may have

interacted with methyl parathion resulting in fluorescence quenching. Further experiments,

including ITC based studies will further confirm the binding of methyl parathion with

GFPuv.

Table 6.1: Organophosphate Binding sites (Y residues in vicinity of positively charged

amino acids) are shown as Y* within respective motifs of human and mouse Transferrin

(Swiss Prot accession # P02787 and Q92111) (Reference: Li et al., 2009).

clxi

OP: Organophosphate compound,

FP-biotin: 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide,

DFP: Didisopropylfluorophosphate,

CPO: Chlorpyrifos oxon,

OP-Tyr: Tyrosine residue at which OP binds

clxii

Fig 6.5: Protein sequence and Ribbon diagram of the backbone of GFPuv (modified from

Khan et al., 2006) showing the chromophore (space filled) and tyrosine residues (shown in

red font in structure and highlighted with cyan color in protein sequence. Positively

charged amino acids are highlighted in yellow color). Most potential binding sites (Y

residues in vicinity of positively charged amino acids) are highlighted in blue boxes.

6.5 Conclusions

>gi|1490529|gb|AAB06048.1| GFPuv

[Cloning vector pGFPuv]

MSKGEELFTGVVPILVELDGDVN

GHKFSVSGEGEGDATYGKLTLK

FICTTGKLPVPWPTLVTTFSYGV

QCFSRYPDHMKRHDFFKSAMPE

GYVQERTISFKDDGNYKTRAEV

KFEGDTLVNRIELKGIDFKEDGNI

LGHKLEYNYNSHNVYITADKQK

NGIKANFKIRHNIEDGSVQLADH

YQQNTPIGDGPVLLPDNHYLSTQ

SALSKDPNEKRDHMVLLEFVTA

AGITHGMDELYK

clxiii

This preliminary proof of principle experiment has identified the potential of

GFPuv purified protein for methyl parathion biosensing applications. The equipment

needed is relatively very simple (sampling tubes, purified GFPuv protein, fluorometer and

UV resistant glasses) which is less costly, easy to use and can be carried to remote areas

very easily where needed. Mutagenesis based further experiments will be helpful in

exploring biosensor variants of GFPuv.

clxiv

7.0 Characterization of a Novel Fluorescent Protein from Hydnophora

rigida with Green Emission

7.1. Introduction

Fluorescent proteins (FPs) are unique biological agents which do not require any

enzymatic modification or cofactors for the formation of functional structure for their

biological applications (Tsien, 1998). Green fluorescent protein (GFP) was originally

isolated from Aequorea jellyfish in 1962 (Tsien, 1998). Since then, numerous genes for

many fluorescent proteins (FPs) have been cloned from natural sources including

hydrozoans and anthozoans (Prasher, 1992; Matz et al., 1999; Frommer et al., 2009). A

broad spectral diversity of GFP-like proteins occurs in coral proteins (Labas et al., 2002;

Verkhusha and Lukyanov, 2004). Availability of a diverse collection of FPs along with

newly identified novel FPs provides an ease of wide range of choices of using them in

biotechnology for biosensing, labeling of cells and cell organelles, making fusion proteins

for in vivo and in vitro studies (Gonzalez and Bejarano, 2000) and to track the behavior of

molecules of interest in vivo (Chudakov et al. 2003; Verkhusha and Sorkin 2005). During

the current study the newly identified novel protein from Hydnophora rigida called as

HriGFP (Accession: ABZ11026) (Bokhari et al., unpublished work) was further

characterized by studying its expression, purification, sequence analysis and structure

modeling.

clxv


7.2.1 HriGFP expression and purification

The open reading frame of HriGFP gene was chemically synthesized (GenScript,

USA) and cloned into pET28a+ vector with addition of N-terminal His tag and transformed

into E. coli BL21 (DE3) cells. The transformed bacterial cells were incubated overnight

(37 oC) in agar plates containing kanamycin (25 mg/mL). Glycerol stocks were made from

a single isolated colony. Several small scale expression experiments were carried out for

optimizing the protocol for higher expression of HriGFP protein. The previously described

method was used (Bokhari et al., 2010) with appropriate modifications for large scale

expression. Cell pellets expressing HriGFP (induced with 0.15 mM IPTG in the presence

of 25 mg/ml kanamycin) were collected by centrifuging at 6000 rpm (JLA 8.1000 fixed

Angle Rotor, Beckman Coulter) and resuspended in 50 ml of lyses buffer (50 mM Tris pH

8.0, 400 mM NaCl, 5% (v/v) glycerol, TritonX 100, 5 mM Imidazole, 1 mM PMSF).

Bacterial cells were lysed by sonication and centrifugation of the lysate was done for 30

minutes at 18,000 rpm (JA-20 Fixed Angle Rotor, Beckman Coulter) at 4 oC. Resulting

supernatant was mixed with Ni+2-NTA (Qiagen) resin (2 mL), (pre-equilibrated with lyses

buffer) and placed for binding at 4 oC for 1 hour. After binding step, the Ni+2-NTA resin

was washed thrice with 10 mL binding buffer containing 30 mM imidazole. Elution of

HriGFP was done with 10 mL elution buffer containing 600 mM Imidazole. Eluted

fractions were analyzed on 12% SDS PAGE gel. The eluted protein from Ni+2-NTA affinity

chromatography was further purified through the HiloadTM 16/60 SuperdexTM prep grade

(Amersham Biosciences, Sweden) pre-equilibrated in a gel filtration column with a buffer

containing 20 mM Tris (pH = 8.0), 100 mM NaCl and 5% (v/v) glycerol. All the

chromatographic purification was performed at 4 oC using the AKTA FPLC.

7.2.2 Dynamic light scattering (DLS)

clxvi

DLS experiment was performed by using a DynaPro instrument (Protein Solutions,

USA) to analyze the homogeneity of fractions eluted from gel filtration column with

various protein concentrations. The hydrodynamic parameters of HriGFP were calculated

by the software provided by the manufacturer.

7.2.3 Peptide Mass Finger print (PMF)

Coomassie-stained SDS-PAGE band containing pure HriGFP was subjected to the

peptide mass finger print experiment in order to confirm its identity. The steps involved in

peptide mass fingerprint were as follows:

(1) In-gel reduction/alkylation,

(2) In-gel digestion,

(3) Sample cleanup and concentration

(4) MS and MS/MS sequencing of the protein digest.

(5) Proteins were identified using Mascot program

(http://www.matrixscience.com).

7.2.4 Searching for similar proteins

The online tools such as NCBI BLAST and Cobalt RID (seq) were used to identify

the homologous protein sequences of HriGFP. The sequence alignment was performed

with the key homologues to identify the conserved motif. The Aequorea victoria (AviGFP),

Montastrea cavernosa (McaGFP) and Hydnophora rigida (HriCFP and HriGFP)

sequences were aligned using CLUSTALW. Phylognetic tree was generated by neighbor

joining tree drawing method by using the bioinformatics tool MEGA5.

7.2.5 Identification of fluorophore

http://www.matrixscience.com/

clxvii

HriGFP protein sequence was compared with other FPs in order to identify HriGFP

fluorophore. The Horn coral fluorescent protein sequences were compared with HriGFP

and HriCFP protein sequences to identify a fluorophore motif of HriGFP and HriCFP

corresponding to the QYG and SYG fluorophore motifs of the Aequorea victoria and

Montastrea cavernosa sequences. Manual positioning of sequences and ClustalW were

used for the sequence comparisons.

7.2.6 Structure Prediction on the basis of similar templates

The structure of HriGFP was modeled on Phyre 2 (Protein Homology/Analogy

Recognition Engine) threading server in the intensive mode for fold–recognition and model

building. HriGFP structure was modeled on multiple templates with a confidence value of

up to 90.3%. Seventy percent of residues of HriGFP were modeled at > 90% confidence

level. Templates are shown in Table 7.1. HriGFP structure is shown in Fig 7.7 and 7.8 in

results section.

clxviii

Table 7.1: Details of multiple templates used for prediction of 3D structure of HriGFP by using

Homology modeling method with the help of online tool (Phyre 2) in intensive mode. HriGFP

structure is shown in Fig 7.7 and 7.8 in results section

clxxi

7.3. Results

7.3.1 HriGFP expression and purification

The best conditions for maximum yield of HriGFP in E. coli were incubation at 16

oC temperature with 0.15 mM IPTG for 16 hours which is the same as described previously

for HriCFP (Bokhari et al., 2010). It has been noticed that HriGFP was not completely

trapped in the Ni2+-NTA beads and it was eluted with 30 mM imidazole. The SDS gel of

the protein eluted from the Ni2+-NTA beads showed some impurities (Fig 7.1A) and

subsequently a gel filtration chromatography was used to improve the homogeneity of

HriGFP (Fig. 7.1B). The gel filtration profile suggests that HriGFP elutes as the monomer

(Fig 7.2).

A B

Fig. 7.1: HriGFP purification

(A) HriGFP expression and purification test in 12% SDS-PAGE. Lane M: protein marker,

Lane 1: Cell lysate, Lane 2: Soluble, and Lane 3: Insoluble fractions, Lane 4: Ni NTA

purification of HriGFP, Lane 5: Flow through after binding with Ni NTA, Lanes 6, 7:

clxxii

Washes with 30mM imidazole in lyses buffer to remove the non specifically bound protein

to the beads, Lane 8: Elution

(B) Lane 1: marker, Lane 2: HriGFP purified by gel filtration chromatography

Fig. 7.2: Gel filtration chromatography of HriGFP. Elution profile shows that HriGFP

elutes as the monomer.

7.3.2 The Dynamic Light Scattering (DLS)

The DLS experiments revealed that the purified HriGFP was a homogenous species

in solution at relatively low protein concentration (<6 mg/ml). However at higher

concentration (≥8 mg/ml) the DLS experiments showed the presence of aggregated HriGFP

for the used conditions.

7.3.3 The peptide mass fingerprint

Peak of purified

HriGFP

clxxiii

The peptide mass fingerprint results (Fig 7.3) confirmed the identity of HriGFP.

Search in primary sequence data base (NCBI) by using the Mascot search engine (Matrix

Science) showed the highest score with HriGFP.

A

B

MPRISDKLMKTRWRGFHSIPSIPPDLGGIYGIGEKTSRRKTTEHLYTGRAKDIKSRLMK

HKYGHQAIDRKIRSNIKQKKLSDLRFKFVEERNHKAKEGLAIEGLKKKLGYAPRFNLRK

RGWIKAKEKPRPQKEG

Fig. 7.3: (A): Mass spectrum of tryptic peptides digest of HriGFP. Spectra were recorded

in a MALDI TOF/TOF in reflection mode. MS/MS of selected peptides were determined.

The protein identity was established by a mass fingerprint search in primary sequence

database (NCBI) using the Mascot search engine. (B) Tryptic digested peptide that shows

match with HriGFP protein in NCBI database as shown in bold underline.

7.3.4. Sequence analysis Results

799.0 1441.8 2084.6 2727.4 3370.2 4013.0

Mass (m/z)

6292.5

0

10

20

30

40

50

60

70

80

90

100

Final - Shots 1000 - 120214_26; Run #1; Label B1

2154.0779(R13700,S1807)

1077.5226(R11753,S532)1993.9437(R13236,S593)

959.4601(R10693,S352)2222.0781(R14378,S331)1475.7335(R12527,S280)879.5040(R10541,S236)

1307.6659(R12104,S163)1699.8120(R13759,S147)2211.0662(R13049,S138)1205.6133(R12391,S58)929.5140(R9774,S44) 2555.2842(R13973,S57)1657.7687(R11902,S35)

clxxiv

HriGFP protein sequence was analyzed for similarities with other proteins using

online tools (NCBI BLAST, and Cobalt RID (seq) to search for any similar proteins. In

addition HriGFP amino acid sequence was searched against the protein databank (PDB) to

identify the presence of any similar protein structures. Results of multiple sequence

alignments of protein sequences using NCBI BLAST and Cobalt RID (Seqs) indicated that

HriGFP has highest similarity with HriCFP (90%) followed by Hypothetical protein ACD

(GenBank Acc: EKD33383.1) (50%), Putative uncharacterized protein (CCY00305.1)

(42%), Hypothetical protein (YP_007918.1) (30%), UvrABC system protein C

(YP_004446894.1) (31%), UvrABC system protein C (YP_004253888.1) (30%),

Excinuclease ABC subunit C (YP_005508972.1) (30%), Excinuclease ABC subunit C

(YP_003389618.1) (30%), uvrABC system protein C (27%), Excinuclease ABC subunit C

(WP_009137569.1) (27%), hypothetical protein (WP_019894762.1) (26%). However

UvrABC system protein C (Table 7.1 in Materials and Methods section) showed the

highest similarity when sequence similarity was searched against PDB databank. Phyre 2

in intensive mode also indicated UvrABC system protein C as the best available template

for homology modeling. Sequence similarities are shown in Fig 7.4

clxxv

Fig. 7.4: Sequence similarity of HriGFP with other proteins. Similar residues are shown in

red letters. Accession numbers of respective proteins are given in blue letters on left hand

side.

7.3.5. Phylogenetic tree

It is apparent from phylogenetic trees that H. rigida fluorescent proteins (HriGFP

and HriCFP) are phylogenetically unrelated to any other FP. In both trees it makes a distinct

out-group and branch length of about 100 timelines unit shows that there is only little or

no similarity between the H. rigida FPs and other anthozoan FPs at both gene and protein

level.

clxxvi

Fig. 7.5: Neighbor joining tree of Anthozoan fluorescent proteins using nucleotide

sequences of genes.

clxxvii

Fig. 7.6: Neighbor joining tree of Anthozoan fluorescent proteins using amino acid

sequences.

clxxviii

7.3.6. Homology modeling: prediction of 3D structure

Protein modeling by using Phyre 2 resulted in a 3D structure of HriGFP which

lacked beta barrels observed in other FPs. The predicted structure of HriGFP comprised of

three-stranded β sheets (β1↑, β2↓, β3↑) flanked by one α helix (α1) at N terminus and three

α helices (α4, α5, α6) at C terminus while α2 and α3 occurred between β2↓ and β3↑.

Putative fluorophore is at C terminus of α2. Seventy percent of the residues were modeled

at > 90% confidence.

clxxix

Fig. 7.7: The predicted structure of HriGFP by using Phyre 2 in intensive mode.

7.3.7. Fluorophore identification

The fluorophore motif was found to be sited in the outer part of the protein (located

at C terminus of an α helix (α2)). The location illustrated hydrophilic nature of fluorophore

which is totally opposite as compared to other Anthozoan fluorescence proteins (which

have their fluorophore located in the internal core surrounded by beta barrel).

A B

Fig. 7.8: Location of fluorophore residues KYG in HriGFP structure, K is the part of α2,

while Y and G residues are located next to K residue between α2 and α3.

clxxx

7.4. Discussions

Previously several distinct FPs with different spectral properties have been isolated

from a single Anthozoa species (Kelmanson and Matz, 2003; Kao et al., 2007). Similarly,

both HriCFP (Bokhari et al., 2010) and HriGFP isolated from H. rigida have less sequence

similarity with other known FPs due to which these two proteins are phylogenetically

distinct from other FPs.

It is interesting to understand the structure of a protein in order to decode the role

of various types of structures in protein function. Although wild type GFP of A. victoria

and most of other FPs have a beta barrel structure, however recently solved protein

structures of some of FPs have shown absence of beta barrel, although such proteins are

very few. Some examples of FPs without beta barrel structure include UnaG (PDB ID:

413B) a fluorescent protein from muscles of Japanese eel (Kumagai et al., 2013), BFPvvD8

(PDB ID: 3P19) which is an NADPH dependant blue FP from Vibrio vunificus CKM-1

(Kao et al., 2011), TagRFP fluorescent protein (PDB ID: 3M22) an artificial gene

(synthetic construct expressed in E. coli) (unpublished results of Malashkevich et al.),

green fluorescent protein (PDB ID: 2PSF) from Renilla reniformis (Loening et al., 2007),

a small sized (115 amino acid residues) fluorescent protein called iLOV (PDB ID: 4EES)

(Christie et al., 2012) and a GFP like protein called copGFP (PDB ID: 2G3O) from

copepod (Wilmann et al., 2006). These non-beta barrel FPs alongwith our non-beta barrel

structure of HriGFP suggest that proteins can exhibit fluorescent properties even in the

absence of a beta barrel structure. These alternative structures may prove to be valuable

clxxxi

guiding models for building small sized artificial FPs for biotechnology applications.

Interestingly, the homology based predicted structure of HriGFP closely resembled the

known structure of iLOV (Fig. 7.9) although Phyre2 program in intensive mode did not

include iLOV as a template due to absence of significant similarity between the amino acid

sequences of iLOV and HriGFP.

Fluorophore of FPs is responsible for their fluorescence. Fluorophore is formed

spontaneously from an indigenous tri-peptide motif of FPs. The reported fluorophore in

the M. cavernosa GFP is a QYG motif (Tsien, 1998) whereas amino acid sequence

comparisons indicated a putative KYG fluorophore motif for HriGFP which is the same as

for HriCFP (Bokhari et al., 2010).

A: HriGFP (green fluorescent protein of H. rigida) (This study)

B: iLOV (FMN binding fluorescent protein) (Christie et al., 2012)

clxxxii

Fig. 7.9: Comparison of protein structure of HriGFP and iLOV. (A): The structure of

HriGFP predicted by using Phyre 2 in intensive mode. The HriGFP structure is strikingly

similar to the structure of Arabidopsis photoprotein iLOV (PDB ID: 4EES) (Fig. B)

(Christie et al., 2012).

The fluorescence excitation and emission maxima of HriGFP (507 and 527 nm,

respectively) were unique, when compared to those of 490 and 509 nm for enhanced GFP

(GFPmut1) (Cormack et al., 1996), 395 nm and 508 nm of wild type GFP and 450 nm and

495 nm for HriCFP (Tsien, 1998; Bokhari et al., 2010). HriGFP structure revealed that its

putative fluorophore motif (KYG) was found to be sited in the outer part of the protein

(located at C terminus of an α helix (α2)). The location illustrated hydrophilic nature of

fluorophore which is totally opposite as compared to the fluorescence proteins bearing beta

barrel structures (which have their fluorophore located in the internal core surrounded by

beta barrel).

It is previously noted that FMN (flavin mononucleotide) binding FPs produce cyan

to green fluorescence (Drepper et al., 2010) which is the same as observed in HriCFP

(cyan) and HriGFP (green). Because FMN is naturally present in all prokaryotic and

eukaryotic cells (Abbas and Sibirny, 2011), so we can not exclude the possibility of

interaction of HriGFP with FMN in studied bacterial host cells. Thus whether FMN has

any role in the fluorescent properties of HriGFP needs to be clarified in future studies.

clxxxiii

FPs from anthozoan and hydrozoans tend to oligomerize and hence are avoided to

be used in constructing fusion proteins whereas engineered monomeric variants can

overcome such problems (Karasawa et al., 2004; Muller-Taubenberger et al., 2006).

However, there have been rare reports of naturally existing monomeric FPs (Karasawa et

al., 2004; Shagin et al., 2004; Gurskaya et al., 2006). Being a monomer FP, HriGFP like

HriCFP (Bokhari et al., 2010) is small enough to tag proteins without affecting either

folding or function to monitor cellular processes and interactions in cells. In many cases

the fluorescent marker protein needs to be monomeric for correct localization of the fusion

partner (Shaner et al., 2004; Merzlyak et al., 2007).

A fluorescent protein smaller than GFP (238 amino acids) will improve the labeling

of peptide-fused recombinant proteins in living cells (Wang and Hazelrigg, 1994) and it is

also advantageous for the development of virus-based vectors (Baulcombe et al., 1995).

Because of the small size, HriGFP is less likely to affect the tagged protein's biochemical

properties and functions. Small fluorescent protein tags such as HriGFP and HriCFP

(Bokhari et al., 2010) may also be superior over larger proteins because they would have a

minimal impact on host metabolism allowing more resources to be committed to the

production of the recombinant target protein (Siepert et al., 2012). The relatively small size

of the protein may facilitate its diffusion throughout the cytoplasm of extensively branched

cells like neurons and glia where the diffusion of large proteins would be difficult. A

relatively small size (≈150 amino acids) is therefore a desirable feature for generating

fluorescently labeled fusion proteins with small imprints (Mukherjee et al., 2012). The

small size of H. rigida fluorescent proteins (HriCFP and HriGFP), along with their

distinguishable excitation and emission wavelengths, may make them ideal candidates for

both imaging and non-imaging uses to determine cellular protein tracking and localization.

Our results indicate that HriGFP is part of non-beta barrel FPs family more closely

related to iLOV and related FMN binding FPs. However sequence of HriGFP did not show

high/significant similarity with any of the known FPs, including iLOV. Thus HriGFP is a

novel green fluorescent protein which provides opportunity of smaller fluorescent

molecules as compared to most of other previously known FPs. As HriGFP predicted

structure is similar to iLOV, so it will be important to find out any similarities of functions

clxxxiv

between HriGFP and LOV domains in order to understand the physiological functions of

HriGFP in corals.

7.5. Conclusions

Our results (monomeric FP with hydrophilic fluorophore and non-beta barrel

structure of molecule, phylogenetic distinction from other FPs) indicate that HriGFP is a

novel green fluorescent protein which provides us opportunity of smaller fluorescent

molecules as compared to most of other previously known FPs. HriGFP may have potential

application as a reporter protein in future biosensing studies and biotechnology.

clxxxv

8.0. Methyl Parathion Biodegradation: Isolation and Characterization of

Methyl Parathion Utilizing Bacterial Isolates for Bioremediation

Applications

8.1. Introduction

Extensive use of pesticides has raised new challenges for mankind. Methyl

parathion is one of the highly toxic organophosphate pesticides affecting non-targeted life

(Shimazu et al., 2001). Contamination of environment (water, soil and air) with methyl

parathion and other pesticides has been reported in various agricultural areas around the

globe. Methyl parathion can even be detected in some sources of drinking water (for

example Rawal Lake of Islamabad, Pakistan (Ahad et al., 2006; Tariq et al., 2007).

Organophosphate pesticides (OPs) may produce a wide range of toxic effects and even

death of humans and other species. Some studies reported cytogenetic and DNA damage

cases from agricultural areas of Pakistan (Multan, Bahawalpur, Rahim Yar Khan etc.) in

workers exposed to pesticides (Bhalli et al., 2006; Ali et al., 2008; Bhalli et al., 2009).

Degradation of methyl parathion helps to reduce the mammalian toxicity (Gilbert

et al., 2003). Bacterial population isolated from the polluted soils may have the ability to

degrade methyl parathion thus providing novel and economic catabolic strategies for rapid

and safe disposal of pesticides (Arfmann et al., 1997; Arfmann et al., 1997; Gilbert et al.,

2003; Zhang et al.2005). Previously several studies have reported methyl parathion

degrading bacterial isolates, however little is known about their combined tolerance to

metals and antimicrobials in addition to utilization of methyl parathion. This chapter

describes the results of combined tolerance against metals and antibiotics in methyl

parathion utilizing bacteria which may help them to survive in multiple contaminated areas

for bioremediation applications.

Methyl parathion utilizing bacterial isolates may help in bioremediation (Ma et al.,

clxxxvi

2001; Shimazu et al., 2001; Zhang et al., 2005). However their survival in the

decontaminated soil (after their application in the field) is very less studied. Vulnerable

bacterial species of the biodegrading consortium may need supportive measures for their

prolonged survival. Sol gels are hybrid organic-inorganic compounds (Livage, 1997)

which are particularly useful for the immobilization of cells because of many supportive

features including their rigidity and thermal and structural stability. Cell immobilization

consists of restricting cellular mobility within a defined space while retaining catalytic

activity and exchange of chemicals with the environment and signal transduction (Fennouh

et al., 2000; Premkumar et al., 2002 a, b; Yu et al., 2005). Previously, generic studies for

testing the bacterial survival in sol gel has been carried out mostly in E. coli (Fennouh et

al., 2000; Premkumar et al., 2002(a); Premkumar et al., 2002(b); Yu et al., 2005; Alvarez

et al., 2007; Soltmann et al., 2008). It has been noticed that bacterial survival is better in

sol-gel as compared to aqueous suspension (Yu et al., 2005). However the goal of enhanced

duration of bacterial survival in sol gel is still not fully addressed. Bacteria remain isolated

in sol gel matrix instead of forming colonies or aggregates (Nadine et al., 2002) which can

be very important for enhancing survival of a consortium of unrelated species in the same

environment. The silicate cages prevent both escape and proliferation of the entrapped

bacteria, but allow free sample diffusion (Premkumar et al., 2002(a); Premkumar et al.,

2002(b); Fesenko et al., 2005). Thus it is expected that sol-gel entrapment would support

the co-existence of unrelated bacterial species, which may otherwise inhibit the growth /

activity of each other.

In order to extend the existing knowledge of cell survival in sol gel matrices, we

report here the survival of a methyl parathion utilizing bacterial consortium in

tetraethoxysilane (TEOS) and sodium silicate based sol gel with some modifications from

the previously reported storage conditions of sol gel immobilized bacteria.

clxxxvii

8.2. Materials and methods

8.2.1. Soil Samples for bacterial isolation

Five soil samples were taken randomly from each sampling site from 12 highly

contaminated agricultural areas across Pakistan (Fig 8.1- 8.2, Table 8.1). Pesticides are

extensively used in these areas and water and soil samples have been reported to be

contaminated with methyl parathion and other pesticides. Samples were taken from the top

15 cm of soil, sieved through a 2 mm pore size sieve and kept at 4 oC prior to use. Soil

samples with no previous history of pesticide use were included as control.

clxxxviii

Fig. 8.1: Soil sampling sites are indicated by red circles in the map of Pakistan

(Contaminations of these sites with methyl parathion are reported in previous studies: Ahad

et al., 2000; Tariq et al., 2007; Ahad et al., 2006; Nafees et al., 2008)

clxxxix

cxc

Fig. 8.2: Pictorial view of some of sampled fields

Table 8.1: Levels of methyl parathion contamination reported at sampling location

Sampling

site Location

Methyl

parathion

conc.

Sample Reference

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

Swabi

Chota Lahore

Mardan

Takhtbhai

Islamabad

Bahawalpur

Muzaffargarh

D.G khan

Rajanpur

Swat

Malakund

Lower Dir

0.06 µg/L

0.01 µg/L

0.05 µg/L

0.01 µg/L

0.38 µg/L

2.5 µg/L

2.5 µg/L

2.5 µg/L

2.5 µg/L

128 mg/kg

128 mg/kg

128 mg/kg

Water

Water

Water

Water

Water

Water

Water

Water

Water

Soil

Soil

Soil

Ahad et al., 2000

Ahad et al., 2000

Ahad et al., 2000

Ahad et al., 2000

Ahad et al., 2006

Tariq et al., 2004

Tariq et al., 2004

Tariq et al., 2004

Tariq et al., 2004

Nafees et al., 2008

Nafees et al., 2008

Nafees et al., 2008

cxci

8.2.2. Isolation of Methyl parathion utilizing bacteria

Bacterial strains were cultured from collected soil samples by using methods of Chaudhry

et al. (1988) and screened for methyl parathion hydrolase activity by methods of Cui et al.

(2004), with some modifications. Briefly, each of 10 g soil sample was suspended in 30

mL of MSM (NH4NO3, 1 g; MgSO4·7H2O, 0.2 g; Ca(NO3)2·4H2O, 0.04 g; and Fe2(SO4)3,

0.001 g per liter) (Chaudhry et al. 1988) supplemented with 5 mg of methyl parathion in a

250 mL flask and incubated at 28 °C (pH 7.0) on a shaker for seven days. Resultant mid

log cultures (OD = 0.6; 2.2 × 108 CFU mL−1) were used to inoculate fresh 20 mL of MSM

supplemented with methyl parathion. The resultant mid log cultures were re-inoculated and

grown in fresh media as described above and the process was repeated for a total period of

5 weeks. Methyl parathion concentration was gradually raised to 1 mg/mL during 7 similar

subsequent transfers. The ability of the bacterial cultures to degrade methyl parathion was

routinely checked visually for the disappearance of methyl parathion crystals from the

bottom of culture medium with a simultaneous increase in optical density as an indicator

of substrate utilization accompanied by bacterial growth (and was subsequently confirmed

by spectrophotometric analyses for methyl parathion hydrolase activity) (Cui et al., 2004).

The resultant mid log cultures (OD = 0.6; 2.2 × 108 CFU mL−1) were transferred into fresh

MSM supplemented with methyl parathion as the sole source of carbon. Methyl parathion

degradation was quantified by methyl parathion hydrolase assay. Cultures which

consistently degraded methyl parathion in 5 subsequent transfers were maintained in

autoclaved liquid MSM supplemented with methyl parathion (1 mg/mL). The methyl

parathion resistant bacteria surviving in the media were isolated for further screening.

Uninoculated controls were used at every step during the experiment as control.

8.2.3. Selection of pure bacterial colonies

Pure colonies were isolated from resultant bacterial cultures by conventional

streaking method. The mid log bacterial cultures with equal number of CFUs/mL were spot

inoculated on plates containing MSM/LB agar, supplemented with 100 µg/mL of methyl

parathion. The resultant colonies with yellow zones of p-nitrophenol around them were

further streaked in two subsequent steps on the similar media to get the purified isolated

cxcii

cultures. The diameters of the yellow zones of p-nitrophenol around the colonies were

recorded (Cui et al., 2004). Ability of these pure cultures to degrade methyl parathion (28

oC, pH 7.0) was subsequently determined by methyl parathion hydrolase assay.

8.2.4. Identification of isolates

Bacterial strains were identified by conventional methods (colony and cell

morphology, biochemical characterization) and additional confirmation of the

identification was done by PCR amplification and sequencing of 16S rDNA segments.

DNA of bacterial strains was extracted by using phenol chloroform method (Cheng and

Jiang, 2006). PCR amplification of 16S rDNA was done by using primer set described by

Hertogh et al., 2006. Fifty μL reaction mixtures containing 5 μL DNA templates, 50 pmol

of each primer, 1.5 mM MgCl2, 0.2 mM of dNTPs and 2.5 U Taq DNA polymerase

(Fermentas) was used for PCR amplification. Thermal cycle reaction conditions were as

follows: 94 oC for 5 min; 30 cycles of denaturation at 94 oC for 40 seconds, annealing at

56 oC for 40 seconds and extension at 72 oC for 1 min. A final extension of 72 oC was run

for 7 min. PCR products were purified by PCR purification kit (Qiagen) and purified

products were sequenced by Macrogen, Korea. The sequences were compared with 16S

rDNA sequences available at the public database of GenBank (using the BlastN program)

for identification of strains.

8.2.5. Methyl parathion Hydrolase assay

Methyl parathion hydrolase activity of purified bacterial isolates was determined

by methods of Cui et al. (2004). Bacterial isolates were allowed to grow in liquid cultures

for 24 hours. Bacterial cells were harvested from cultures by centrifuging at 15,000 × g for

10 minutes at 4 °C. Bacterial cells were washed twice in 20 mM sodium phosphate buffer

(pH 7.5) and centrifuged again as above. The cell pellet was resuspended in PBS, and cells

were disrupted by sonicating them 15 times for 30 seconds periods at 15 seconds intervals

in ice container. Unbroken cells and cellular debris were removed by centrifugation

cxciii

(15,000 × g for 30 minutes at 4 °C). The clear homogenous supernatant was used to

determine methyl parathion hydrolase activity. Methyl parathion hydrolase activity was

determined at 35 °C by measuring the increase in absorbance at 400 nm resulting from the

p-nitrophenol released by the hydrolysis of methyl parathion. Enzyme samples (20 μL

supernatant) were added to an assay mixture containing 974 μL 200 mM phosphate-

buffered saline (PBS, pH 8) and 6 μL methyl parathion (10 mg/mL). Samples (20 µL

supernatant) boiled for 5 minutes were used as controls. One unit (U) of methyl parathion

hydrolase activity was defined as the amount of enzyme required to hydrolyze 1 μmol

methyl parathion in 1 min at 35 ºC as described previously (Chaudhry et al., 1988, Cui et

al., 2004). Enzyme activities present in 20 µL samples were determined in triplicate and

compared.

8.2.6. Heavy Metal tolerance

Briefly, 0.1 mL of bacterial cultures having 0.5 OD were aseptically inoculated on

MSM supplemented with methyl parathion as sole source of carbon and increasing

concentrations of the following heavy metals: As-III, As-V, Cd2+ and Hg2+. Bacterial

growth was observed during 24-48 hours. Strains growing in the presence of metals were

recorded. MTC (maximum tolerance concentration) was recorded as the highest

concentration of metal which did not affect the viable count of bacteria and allowed growth

after a period of two days (48 hours) (Ganiyu et al., 2010). Media prepared and inoculated

without metal and Non-inoculated media supplemented with metals were used as controls.

Test was done in triplicate

8.2.7. Antibiotic resistance

Susceptibility to commonly used antibiotics was determined using standard disc

diffusion method in accordance with Clinical and Laboratory Standard Institute

recommendations (CLSI, 2006). Antimicrobial discs (Oxoid, UK) included Ciprofloxacin

(CIP: 10 μg), Nalidixic acid (NAL: 30 μg), Ceftazidime (CAZ: 30 μg),

cxciv

Sulphamethoxazole-Trimethoprim combination (SXT: 25 μg), Tetracycline (TE: 30 μg),

Chloramphenicol (C: 30 μg), Erythromycin (E: 15 μg), Gentamicin (CN: 10 μg) and

Ampicillin (AMP: 25 μg).

8.2.8. PCR for integrons and antimicrobial resistance genes

Total genomic DNA templates of bacterial strains were extracted by Phenol-

Chloroform extraction method. Sulphamethoxazole resistance determinants sul1 and sul2

were identified by PCR amplification using gene specific primers (Invitrogen, Carlsbad,

USA). The amplification of sul1 was performed using the forward primer sul1F and reverse

primer sul1R. Amplification of sul2 was done by using the forward primer sul2F and the

reverse primer sul2R (Blahna et al., 2006). Plasmids (extracted by using Miniprep (Qiagen)

plasmid extraction kit) were used as templates to amplify internal fragments of the

quinolone resistance genes qnrA (580 bp), qnrB (264 bp) and qnrS (428 bp) (Cattoir et al.,

2007). Primer details are given in table 8.2.

Table 8.2: Primers used for amplification of antibiotic resistance gene segments

Gene of

interest

Primer

name


size

Sul1 sul1F CTTCGATGAGAGCCGGCGGC

55 oC 417 bp

sul1R GCAAGGCGGAAACCCGCGCC

Sul2 sul2F GCGCTCAAGGCAGATGGCATT

55 oC 285 bp

sul2R GCGTTTGATACCGGCACCCGT

QnrA qnrAF AGAGGATTTCTCACGCCAGG

55 oC 580 bp

qnrAR CCAGGCACAGATCTTGAC

QnrB qnrBF GGGTATGGATATTATTGATAAAG 55 oC 264 bp

cxcv

qnrBR CTAATCCGGCAGCACTATTA

QnrS qnrSF GCAAGTTCATTGAACAGGGT

55 oC 428 bp

qnrSR TCTAAACCGTCGAGTTCGGC

TA: Annealing Temperature for PCR amplification

Screening for the presence of class 1, 2 and 3 integrons (int1, int2, int3) was done

with the help of PCR-RFLP method (White et al., 2001). The sequences of the degenerate

primers to hybridize to conserved regions of integron encoded integrase genes. Integrase

PCR products were digested using Hinf1 restriction enzyme according to the

manufacturer’s instructions (Fermentas). Classification of integrons was done on the basis

of the size of restricted fragments after treatment of hep amplified products with Hinf1 (491

bp fragment for class 1 integrons, 191 bp and 300 bp fragments for class 2 integrons, 119

bp and 372 bp fragments for class 3 integrons) (White et al., 2001). Primer details are given

in Table 8.3. PCR amplification conditions are given in Table 8.4, 8.5.

Table 8.3: Primers used for PCR-RFLP based identification of integrons (int1, int2, int3)

Primer

name


size

Hinf1 treated

PCR product

Hep 35

TGCGGGTYAARGATBTKGATTT

55 oC 491 bp

int1: 491 bp

int2: 191 bp &

300 bp

hep36 CARCACATGCGTRTARAT

cxcvi

int3: 119 bp &

372 bp

B = C or G or T; K = G or T; R = A or G; and Y = C or T.

TA: Annealing Temperature for PCR amplification

Table 8.4: PCR conditions for amplification of antibiotic resistance genes and integrons

Components Concentration

dNTP

PCR buffer

MgCl2

Each primer (forward/reverse)

Taq. Polymerase

Genomic DNA

0.3 mM

1x (10 mM Tris–HCl pH 9.0, 50 mM KCl)

2.0 mM

0.5 mM

1.5 U

50 ng

Table 8.5: Thermal cycling conditions for amplification of antibiotic resistance genes and

integrons.

Step Temperature Time

cxcvii

1. Initial denaturation

2. Denaturing

3. Primer annealing

4. Chain extension

5. Final extension

95 OC

95 OC

55 OC

72 OC (Go to step 2, 35 ×)

72 OC

4 min

45 sec

1 min

1 min

10 min

8.2.9. Consortium of Bacterial Isolates

A consortium of 7 bacterial isolates, with highest methyl parathion hydrolase

activity among 64 indigenous isolates, was selected for sol-gel immobilization studies.

8.2.10. Methyl parathion hydrolase activity of Consortium

Methyl parathion hydrolase activity of individual isolates was compared with

consortium of the same isolates with the method described in section 8.2.5.

8.2.11. Growth pattern of Consortium members

The growth pattern of bacterial strains was monitored at 28 oC (pH = 7.0) by using

minimal medium with 0.6 mM methyl parathion as source of carbon and phosphorus

(Chaudhry et al., 1988). Growth curves of the cultures were monitored by measuring the

cxcviii

optical density at 600 nm after every hour. Uninoculated minimal media and inoculated

minimal media without methyl parathion were used as control.

8.2.12. Cell immobilization and stability in sol-gel

The consortium was immobilized in sol-gel by using two types of materials: TEOS

and sodium silicate. Cell survival and preservation of methyl parathion hydrolase activity

was studied. In addition, E. coli DH5α cells transformed with pGFPuv (Clontech) were

also immobilized in sol gel and their ability to express GFP after immobilization was

observed by fluorescence microscopy (Leica DM4000 M) as an indicator of preservation

of biological activity.

8.2.12.1. Cell immobilization and stability in TEOS derived sol-gel

Selected isolates were individually inoculated in MSM media for 12 hours.

Overnight grown cultures of pure individual strains were diluted with a ratio of 1:20 in

fresh MSM supplemented with methyl parathion (100 μg mL−1) and grown at 28 oC (pH =

7) up to mid log phase (OD = 0.6). Equal volumes of resultant cultures were mixed together

to develop consortium. The bacterial cells were washed with sterile MSM and concentrated

to yield a suspension of bacterial consortium with 2.1 × 109 CFU mL−1 in fresh media. The

sol was prepared by sonicating a mixture of 1 mL TEOS, 0.2 mL water and 0.06 mL of

0.04 M HCl for 30 min at 20 oC, as described previously (Desimone et al., 2005; Soltmann

and Bottcher, 2008). After addition of 2 mL water, excess ethanol was removed under N2.

A bacterial suspension of consortium (2.1 × 109 CFU mL−1) was mixed with an equal

volume of the sol solution. The solution was left for 2 min for gelation. After gelation, sol-

gels were washed ten times with fresh PBS to remove any residual ethanol and kept in

closed tubes either in MSM containing methyl parathion or in PBS (Dancil et al., 1999;

Desimone et al., 2005). The images of sol-gel immobilized bacteria were obtained by using

30 KV scanning electron microscope (JSM5910, JEOL, Japan). Bacterial cell survival at

different conditions were studied which included the following: (a) suspension of bacterial

cxcix

consortium (2.1 × 109 CFU mL−1) in MSM containing methyl parathion (b) sol-gel

immobilized consortium kept in MSM containing methyl parathion (c) sol-gel immobilized

consortium without MSM and methyl parathion (d) mixture of MSM and methyl parathion

without inoculum, (e) sol-gel without inoculum. The results of bacterial cell survival at

these different conditions were compared.

For determination of viability in suspension, decimal dilutions in LB medium of

the free cell suspensions were plated in duplicate in LB agar and incubated at 37 oC. After

24 h, the number of CFU was determined. For immobilized cell count, an average weight

of 200 mg sol-gel matrix was disrupted using a sterilized glass rod, homogenized in 1 mL

LB medium and used for viable cell counts in the same way as for bacterial suspensions.

8.2.12.2. Cell immobilization and stability in sodium silicate derived sol-gel

One gram of sodium silicate was mixed with 6 mL water and incubated at 80 oC

until complete colloidal suspension was obtained. The sol was allowed to cool to room

temperature and the pH of final mixture was raised to pH = 7 ± 0.02 with 0.75 M citric

acid. Bacterial suspension of consortium (2.1 × 109 CFU mL−1) was mixed with an equal

volume of the prepared colloidal suspension and the solution was left for 2 min to attain

gelation. Bacterial cell survival at different conditions was studied. For viability

determination, an average weight of 200 mg sol-gel matrix (Desimone et al., 2005) was

disrupted using a sterilized glass rod, homogenized in 1 mL LB medium and then two-fold

serial dilutions of the free bacterial suspensions were plated in duplicate in LB agar and

incubated at 37 oC. After 24 hours, the number of CFU mL−1 was determined. All

determinations were made in duplicate for 4 months as was previously reported (Alvarez

et al., 2007). Storage stability in inorganic matrices was studied at 28 oC. Immobilized

consortium in silica matrices (1 mL aliquots) supplemented with methyl parathion (100 μg

mL−1) were stored in labeled 5 mL sealed tubes at 28 oC for 4 months in which the number

of viable cell was analyzed. The numbers of CFU mL−1 were determined by plate count

technique.

cc

8.3. Results

8.3.1 Methyl parathion utilizing bacterial isolates

Sixty four methyl parathion utilizing bacterial strains were isolated from methyl

parathion contaminated soils. These strains were able to grow in the presence of methyl

parathion as the sole source of carbon and consistently degraded methyl parathion during

several sub-culturing steps. Control soil samples did not produce any bacterial growth in

the presence of methyl parathion.

The studied strains produced yellow zones (of p-nitrophenol; 10–21mm diameter)

on MSM agar plates supplemented with methyl parathion. Varying levels of methyl

parathion hydrolase activities (313–675 mU/mL for 64 isolates) were observed via

spectrophotometric analysis. These isolates (n = 64) belonged to the families of

cci

Staphylococcaceae, Vibrionaceae, Moraxellaceae, Pseudomonadaceae and

Enterobacteriaceae. Enterobacteriaceae showed the highest diversity of species. Proteus

was the most prevalent genus (18, (28%)) isolated in this study followed by Pseudomonas

(11, (17%)), Klebsiella (9, (14%)), Escherichia (6, (9%)), Serratia (5, (8%)),

Staphylococcus (4, (6%)), Salmonella (2, (3%)), Enterobacter (2, (3%), Acinetobacter (2,

(3%), Citrobacter (2, (3%), Vibrio (2, (3%)) and Enterobacteriaceae bacterium (1, (1.6%).

(Fig. 8.3)

A selection of 7 bacterial strains with highest methyl parathion hydrolase activity

(enzyme assay ranging from 410 mU mL−1 (for Proteus vulgaris IVTMP1) to 675m U/mL

(for Enterobacter Sp. NII-14)) was used for consortium and sol gel studies.

ccii

Fig. 8.3: Prevalence of various types of methyl parathion utilizing bacterial isolates (n=

64) in soil samples of agricultural regions contaminated with methyl parathion. These

isolates grew in the presence of methyl parathion as sole source of carbon.

8.3.2. Heavy Metal tolerance

14%

9%

8%

17%

6%

28.00%

3%

3%

2%

3%

3%

3%

0% 5% 10% 15% 20% 25% 30% 35%

Klebsiella

Escherichia

Serratia

Pseudomonas

Staphylococcus

Proteus

Salmonella

Enterobacter

Enterobacteriaceae

bacterium

Acinetobacter

Citrobacter

Vibrio

Bac

teri

al i

sola

tes

Prevalence

cciii

Methyl parathion utilizing bacteria in the present study showed high prevalence of

heavy metal tolerance. Order of tolerance to heavy metals in these isolates was as follows:

As-V > Cd2+ > Hg2+ > As-III. Only 4 of 64 methyl parathion resistance isolates did not

show resistance to any of the heavy metal tested. All the remaining 60 of 64 methyl

parathion resistant isolates were also resistant to ≥1 heavy metals tested (Fig 8.4).

Fig. 8.4: Prevalence (%) of heavy metal tolerance in methyl parathion utilizing bacterial

isolates (n = 64)

86%

62%

45%

91%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Cd Hg As-III As-V

Heavy metal tested

Pe

rce

nta

ge

of

res

ista

nt

iso

late

s

cciv

Our results indicate that 55 (86%) of the methyl parathion utilizing isolates (n = 64)

were tolerant to various levels of Cd2+ (MTC range 0.49 to 2.98 mM). Highest metal

resistance was revealed against As-V (58, (91%)) (MTC range 20 to 100 mM), and lowest

against As-III (29, (45%)) (MTC range 2 to 16 mM) in methyl parathion hydrolyzing

bacteria. Forty (62%) of methyl parathion hydrolyzing bacteria showed tolerance to Hg2+.

However MTC ranged from 0.05 to 0.27 mM which indicates relatively lower resistance

to Hg2+ in these strains.

8.3.3. Antibiotic resistance

In the present study, the level of antimicrobial resistance ranged from 40 (62%) (for

NA) to 9 (14%) for CIP. The descending order of resistance to antibiotics was as follows:

NA>TE>SXT>E>AMP>C>CAZ>CN>CIP. Most of the isolates were resistant to multiple

of antibiotics simultaneously. All the methyl parathion utilizing heavy metal resistant

isolates showed resistance to ≥2 (2 to 6) antibiotics tested. Nalidixic acid (40, (62%)) and

Tetracycline (38 (59%)) are the antibiotics to which largest number of isolates were

resistant. These isolates were more sensitive to Ciprofloxacin (9, (14%)), Chloramphenicol

(11, (17%)) and Ceftazidime (13, (20%)) than other drugs to which the sensitivity patterns

were observed (Fig. 8.5).

62%

20%

48%

59%

14%

41%

31%

41%

17%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

NA CAZ SXT TE CIP E C AMP CN

Antibiotics tested

Pe

rce

nta

ge

of

res

ista

nt

iso

late

s

ccv

Fig 8.5: Antibiotic resistance pattern in methyl parathion utilizing bacterial isolates (n = 64). CIP

(Ciprofloxacin), NA (Nalidixic acid), CAZ (Ceftazidime), SXT (Sulphamethoxazole-

trimethoprim), TE (Tetracycline), C (Chloramphenicol), E (Erythromycin), CN (Gentamicin) and

AMP (Ampicillin)

8.3.4. Antimicrobial resistance genes and integrons

PCR results for integron and antibiotic resistance genes were in accordance to the

specific band sizes reported previously for specific genes (Fig. 8.6).

Fig 8.6: Integron and antibiotic resistance genes. M, 100bp DNA Marker; 1, hep amplified

product (491bp); 2, non restricted fragment (hep amplified fragment subjected to Hinf1

restriction enzyme digestion) of int1 (491bp); 3, restricted fragment (hep amplified

fragment subjected to Hinf1 restriction enzyme digestion) of int2 (300bp, 191bp); 4, qnrS

amplification band (428 bp); 5, qnrB amplification band (264bp); 6, qnrA amplification

band (580bp); 7, sul2 amplification band (285 bp); 8, sul1 amplification band (417 bp).

264bp

500bp

100bp

M 1 2 3 4 5 6 7 8 M

491bp 491bp

300bp

191bp

428bp2 580bp

285bp

417bp

264bp

ccvi

Among the 64 isolates, int1 was detected in 25 (39%), int2 in 3 (4.6%), sul1 in 25

(39%), and sul2 was detected in 20 (31%) isolates. Prevalence of qnrS (2 (3%)), qnrA (2

(3%)) and qnrB (5 (7.8%)) was low which correlates with low prevalence of antimicrobial

resistance to CIP (9 (14 %)). Integron 3 (int3) was not detected in any of the studied isolates

(Fig 8.7).

Fig. 8.7: Prevalence (%) of antibiotic resistance genes (sul1, sul2, qnrA, qnrA, qnrB) and

integrons (int1, int2) in methyl parathion utilizing bacteria (n = 64) isolated in this study.

39%

4%

39%

31%

3% 3%

7%

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

int1 int2 sul1 sul2 QnrS QnrA QnrB

Integrons and anbiotic resistance genes

Pre

va

len

ce

(%

)

ccvii

8.3.5. Selection of Bacterial Consortium for sol gel studies:

Short listing of the 64 methyl parathion utilizing bacterial strains (for selection of

consortium and sol-gel stability) was carried out on the basis of (1): the size of yellow zone

around the independent colonies on media containing methyl parathion (100 µg/mL), and

(2): subsequent spectrophotometric determination of methyl parathion hydrolase activities.

Seven Enterobacteriaceae isolates showing highest methyl parathion degrading activities

with enzyme assay ranging from 410 mU/mL (for Proteus vulgaris IVTMP1) to 675

mU/mL (for Enterobacter Sp. NII-14) were selected. The 16 S rDNA of identified strains

showed highest similarities (91 to 99 %) with Proteus vulgaris strain DTQ-LP12.1, Proteus

sp. KRD11, Enterobacter sp. NII-14, Enterobacteriaceae bacterium ASC10, Citrobacter

sp. SR3, Proteus vulgaris strain IVTMP1 and Proteus vulgaris.

Fig 9.1: Cladogram (generated by MEGA 5 on the basis of 16S rDNA sequences) showing

relationship of bacterial species added in consortium.

ccviii

Table 9.1: Identification based on similarities of the 16S rDNA gene (partial sequence) of

methyl parathion degrading bacterial strains with previously known bacterial strains.

Strain

#

Highest homology with 16S rDNA

gene

% similarity NCBI AC #

9

4

6

8

28

12

20

Proteus vulgaris strain DTQ-LP12.1

Proteus sp. KRD11

Enterobacter sp. NII-14

Enterobacteriaceae bacterium ASC10

Citrobacter sp. SR3

Proteus vulgaris strain IVTMP1

Proteus vulgaris

98

94

99

99

91

99

99

EF012763.1

HM345976.1

FJ897480.1

EU040283.1

FJ911682.1

GU361619.1

DQ826507.1

8.3.6. Methyl parathion degrading enzymatic activity of short listed consortium

Methyl parathion degrading enzymatic activity of short listed consortium of seven

strains was compared with the activity of individual isolates and results suggested that the

activity of the consortium was higher (982 mU/ml) than any of individual isolates (which

ranged from 410 to 675 mU/mL).

ccix

Fig. 9.2: Methyl parathion degrading activity of individual bacterial isolates and their

consortium.

8.3.7. Growth pattern of bacterial strains of the consortium

Growth pattern of bacterial strains of the consortium was monitored by using

minimal medium with 0.6 mM methyl parathion as source of carbon and phosphorus. The

growth kinetics of the individual isolates in the presence of methyl parathion revealed

different levels of tolerance to methyl parathion. Proteus sp. KRD11 showed highest (OD

= 2.0 after 12 hours growth) and Enterobacter sp. N II- 14 showed the lowest levels of

tolerance (OD = 1.3 after 12 hours growth) to methyl parathion.

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time (hours)

OD

(6

00

nm

)

Proteus sp. KRD11

Proteus vulgaris strain DTQ-LP12.1

Enterobacteriaceae bacterium ASC10

Citrobacter sp. SR3

Proteus vulgaris strain IVTMP1

Proteus vulgaris

Enterobacter sp.NII-14

ccx

Fig. 9.3: Growth curves of selected individual bacterial strains (solid coloured lines) in

MSM supplemented with methyl parathion (0.6 mM methyl parathion). Dotted coloured

lines represent bacterial isolates inoculated in MSM without methyl parathion. Dotted

black coloured lines represent uninoculated media.

8.3.8. Cell immobilization and stability in sol-gel

Immobilized consortium was stored in sealed tubes at 28 oC (for 4 months) the

number of viable cells and preservation of methyl parathion hydrolase activity was

analyzed during this period. The sol-gel immobilized consortium was studied for stability,

activity and shelf life.

ccxi

Fig. 9.4: Sol-gel with immobilized methyl parathion degrading Bacterial strains (X 8000

magnification)

Viability studies showed that consortium survived the sol-gel immobilization

process. However, post immobilization survival rate was different in case of TEOS and

sodium silicate based sol gel matrices. Our results indicate that the bacterial consortium

cell survival was more uniform in sol-gel when it was supplemented with MSM and methyl

ccxii

parathion either in TEOS (curve B in Fig. 9.5) or sodium citrate based matrices (curve B

in Fig. 9.6) as compared to other storage conditions (curve A and C). Survival of cells in

TEOS based sol-gel without MSM and methyl parathion (curve C in Fig. 9.5) was low with

sharp and continuous fall in the number of surviving cells with the passage of time. Survival

rate was better in case of sodium silicate based sol gel stored with or without MSM and

methyl parathion as compared to TEOS based sol gel.

9.3.4.1 Bacterial survival in TEOS based sol-gel

Fig. 9.5: Stability of methyl parathion degrading consortium in suspension and TEOS

based sol-gel (MSM: Minimal Salt Medium. MP: methyl parathion). (Curve A:

Consortium in suspension with MSM + MP; Curve B: Sol gel immobilized consortium

with MSM + MP

Curve C: Sol gel immobilized consortium without MSM + MP; Curve D: Suspension of

MSM + MP without inoculum; Curve E: Sol-gel without inoculum)

0

2

4

6

8

10

12

14

0 5 10 15

Days

Lo

g C

FU

/ m

l

A

B

C

D

E

Linear

(A)Linear

(B)Linear

(C)

ccxiii

9.3.4.2 Bacterial survival in sodium silicate based sol-gel

Fig. 9.6: Stability of methyl parathion degrading consortium in suspension and sodium

silicate based sol-gel (MSM: Minimal Salt Medium. MP: methyl parathion). (Curve A:

Consortium in suspension with MSM + MP; Curve B: Sol gel immobilized consortium

with MSM + MP Curve C: Sol gel immobilized consortium without MSM + MP; Curve

D: Suspension of MSM + MP without inoculum; Curve E: Sol-gel without inoculum)

0

1

2

3

4

5

6

7

8

9

10

0 20 40 60 80 100 120

Days

Lo

g C

FU

/ m

l

A

B

C

D

E

Linear

(C)Linear

(B)Linear

(A)

ccxiv

8.3.9. GFP expression in sol-gel immobilized cells

In addition to sol gel immobilization of methyl parathion utilizing bacterial

consortium, GFP expression was detected in E. coli DH5α (transformed with pGFPuv) in

sol-gel matrices as an additional marker for detection of biological activity in sol gel

matrices. Sol gel immobilized cells produced fluorescence under UV light due to

production of GFP. Even the fluorescence of GFP was present due to stable nature of GFP

outside the dead cells when the TEOS based gel did not produce any surviving cells in the

TEOS based sol-gel. Results indicate that sol gel immobilized bacterial cells are intact and

biologically active.

Fig. 9.7: Fluorescence microscopy of GFP expressing E. coli DH5α cells immobilized in

sol- gel matrices

ccxv

Light microscopy of the same matrices containing GFP expressing E. coli DH5α

did not produce any fluorescence because fluorescence of GFP can not be detected without

UV and the cell shapes were not obvious under light microscopy due to immobilizing sol

gel layers.

Fig. 9.8: Light Microscopy of sol-gel immobilized GFP expressing bacterial cells (E. coli

DH5α)

ccxvi

8.4. Discussions

Appropriate management of agricultural lands is essential for sustainable

agriculture for fulfilling the basic human needs. Application of heavy metals and pesticides

in industry and agriculture has heavily polluted the exposed environment.

“Bioremediation” is by far the most ideal and most efficient approach for decontaminating

the contaminated agricultural soils (Ahemad and Khan, 2011) because alternative methods

of environmental detoxification are environmentally less friendly and highly costly in

addition to other disadvantages (Strong and Burgess, 2008). Thus methyl parathion

hydrolyzing bacterial strains may be very useful natural tools for recovering the

contaminated areas.

As contaminated ecological niches are the most appropriate sites for isolation of

organisms capable of degrading xenobiotics (Oshiro et al., 1996; Horne et al., 2002), so

we selected methyl parathion contaminated areas for isolation of methyl parathion utilizing

bacteria. Proteus was the most prevalent genus detected in our study. Variations in

geographical positions, weather conditions and soil properties may be one of the reasons

for this variation.

Several of previous studies have reported only one type of methyl parathion

hydrolyzing/degrading bacteria from soil samples (Stenotrophomonas sp. SMSP-1 by Shen

et al., 2010 from China), Pseudomonas putida DLL-1 by Wen et al., 2007 from China,

Ochrobactrum by Qiu et al., 2006 from China, Burkholderia sp. FDS-1 by Zhang et al.,

2006b from China, Pseudomonas diminuta GM by Serdar et al., 1985 from Philipines,

JS018 (related to Roseomonas sp.) by Jiang et al., 2006 from China. JS018 strain was

resistant to ampicillin, penicillin and lincomycin and sensitive to kanamycin, tetracycline

and gentamicin. Only a few studies (including our study) have isolated multiple of methyl

parathion degrading bacteria from contaminated soils. Pino et al., 2011 isolated

ccxvii

Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa, Citrobacter

freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp,

Acinetobacter sp, Klebsiella sp and Proteus sp from Columbia, from a garbage dump site.

These strains were resistant to up to 150 mg/L of methyl parathion. Choi et al., 2009

reported Burkholderia, Arthrobacter, Pseudomonas, Variovorax, and Ensifer from Korea.

Kim et al., 2009 isolated Burkholderia, Pseudomonas, Sphingomonas, Cupriavidus,

Corynebacterium, and Arthrobacter from different contaminated areas soils in Korea.

Twenty-two of 27 isolates in this Korean collection were able to degrade parathion.

The soil habitat is known for its highly diverse nature of inhabitants and more than

10000 species of bacteria may be present in each 1 gram of soil sample (Torsvik et al.

1990, Torsvik et al. 1998). However, only a small proportion of soil bacteria (less than 1

to 10%) are cultivable in lab conditions (Amann et al., 1995; Balestra & Misaghi, 1997;

Dykhuizen, 1998; Janssen et al. 2002). Thus our study (including the previous ones) was

limited to only culturable part of the soil bacterial community. More sophisticated studies

to culture the uncultured bacterial community would give even a better picture of

biodiversity at the environmental level in contaminated agricultural areas.

Organisms living in the contaminated sites not only evolve strategies to cope with

xenobiotic, but also learn to use them to provide energy (Storz and Imlay 1999; Imlay,

2003; Falkowski and Godfrey 2008). Similarly bacteria incidentally exposed to

insecticides evolve mechanisms (bacterial enzymes and pathways) to degrade them so that

they can use them as sources of nutrients to increase their rates of reproduction and

proliferation (Behki and Topp 1993; Chaudhry et al. 2002; Ellis et al. 2001; Yan et al.

2007; Scott et al., 2008; Gao et al. 2010; Khalid et al., 2011). The studied bacterial strains

were able to grow in the presence of methyl parathion as a sole source of carbon. The

studied bacteria might have been able to utilize methyl parathion due to their methyl

parathion hydrolase activity in addition to any other mechanism. Methyl parathion

hydrolase is bacterial enzyme capable of hydrolyzing methyl parathion (Dong et al. 2005)

and its activity is effectively restricted to methyl parathion (Russel et al., 2011). Methyl

parathion hydrolase activity has also been reported previously in strains including

Plesiomonas sp. strain M6, Pseudomonas sp. WBC-3. Three Parathion and Malathion

ccxviii

utilizing bacterial isolates Pseudomonas fluorescens, Bacillus subtilis and Klebsiella sp.

were reported by Karunya and Reetha, 2012. Among the three bacterial isolates, the

bacteria Klebsiella sp. utilized the pesticides effectively and showed maximum growth.

However they did not report heavy metal or antibiotic resistance in these isolates.

At low concentrations, metals can serve important role in enzyme functions (Nies,

1992). Metals are toxic to many species above certain threshold concentrations, but certain

soil bacteria exposed to high levels of toxic metals may evolve to become resistant against

them in order to survive and grow in metal contaminated areas (Elena et al., 2005; Habi

and Daba 2009).

Heavy metal tolerance is an important trait of xenobiotic degrading bacterial

strains. Several heavy metal resistant bacteria are reported with a simultaneous xenobiotic

degrading activity. Sizova et al. (2010) reported two Arsenic resistant rhizospheric

Pseudomonas spp. capable of oxidizing naphthalene. One group in Kuwait reported on

indigenous bacteria with the combined potential for mercury resistance and hydrocarbon

utilization in soils and plant rhizospheres (Sorkhoh et al. 2010a, b; Al-Mailem et al. 2010;

Al-Mailem et al. 2011). Singh et al. 2010 described metal resistance (resistant to lead,

arsenic, cesium, cobalt, nickel, chromium, sensitive to mercury) and antimicrobial

resistance (against lincomycin, penicillin, pefloxacin and cloxacillin) in halotolerant

Bacillus cereus SIU1 strain. However studies on methyl parathion utilizing bacteria did not

address metal tolerance in such bacterial strains. This is the first study conducted to reveal

additional traits of metal tolerance and antimicrobial resistance in methyl parathion

utilizing bacteria.

Majority of methyl parathion utilizing bacteria in the present study were tolerant to

raised concentrations of heavy metals in the presence of methyl parathion as sole source of

carbon which indicates their efficiency to degrade methyl parathion in heavy metal

contaminated areas. The bacterial strains in this study were isolated from pesticide

contaminated areas while it is known that pesticide polluted areas are usually

simultaneously polluted with heavy metals also (Grassi and Netti 2000; Protano et al.

2000). Application of sewage sludge and agrochemicals add a considerable amount of

metals in agricultural soils (Kumar et al., 2011). Heavy metal resistance itself also may

ccxix

have potential applications because bacteria have affinity for metals and bioaccumulation

may help them to decontaminate heavy metals from soil (Rani and Goel 2009). Metal

resistant bacteria are reported to show increased metal uptake when exposed to a raised

metal concentration in the culture media (Ahemad and Malik, 2012). Zolgharnein et al.,

(2010) reported an uptake of metal ions by a Pseudomonas aeruginosa isolate from Persian

Gulf. Intracellular and periplasmic accumulation of heavy metals (cadmium) are reported

in Pseudomonas putida and metal binding and/or efflux mechanisms may be involved in

heavy metal resistance in bacteria (Rani and Goel, 2009), periplasmic accumulation (Naz

et al., 2005), Cytoplasmic accumulation (Yoshida et al., 2002).

Our bacterial isolates were highly resistant against commonly studied heavy metals

(mercury, arsenite, arsenate and cadmium), Comparison of results indicate that these

bacterial strains have the potential to equally exist in a competitive environment with other

metal resistant bacterial isolates of the natural environments for bioremediation from most

of the regions of the world with some exceptions. We found an MTC range of 20-100 mM

for arsenate as compared to previous studies. Similar level of resistance was observed by

Banierrie et al., (MIC = 50 – 125 mM). Kanshik et al., reported a lower level of resistance

(MIC = 23-69 mM), while several studies reported an even higher level of resistance

against arsenate. Cavalca et al 2010 (MTC > 100 mM), Pepi et al., 2007 (MIC = 133.47

mM), Pepi et al., 2011 (MIC = 135 mM). Huang et al., 2010 reported an even higher level

of tolerance (MIC = 400 mM for 12 bacterial isolates).

Our MTC value of 2-16 mM for arsenite resembled tolerance levels of resistant

strains of several studies including Pepi et al., 2011 (MIC = 10 mM), Canalca et al., 2010

(MTC = 10 mM), Pepi et al., 2007 (MIC ≥ 16.88 mM), Cai et al., 2009 ( MIC > 20 mM),

Yoon et al., 2009 (MIC = 26 mM). One study also reported a high variation of 10 – 100

mM MIC value for arsenite (Banerjee et al., 2011).

Our bacterial isolates are resistant to 0.05-0.27 mM mercury which is very high as

compared to some of the studies (0.0067 mM MIC reported by Cabral et al., 2013). On the

other hand some of the studies have also shown a slightly higher level of tolerance to

mercury (Bafana et al., 2010 reported an MTC value of 0.369 mM for mercury).

ccxx

Our isolates showed an MTC values of 0.49-2.98 mM as compared to some higher

levels (MIC = 7 mM reported by Ganesan et al., 2008) and lower levels (MTC = 0.037

mM reported by Bafana et al., 2010). Thus our results combined with literature suggest

that the studied bacterial isolates would be able to co-survive in most of the environments

with highly resistant indigenous environmental bacterial strains. The comparison also

highlights the need to further explore metal resistance in methyl parathion utilizing

bacterial strains from other parts of the world.

In addition to heavy metal tolerance, antibiotic resistance is an additional

supportive tool which bacterial strains may need to survive in highly competitive natural

environments, especially if the contaminated environments. Most of the isolates in the

present study were resistant to multiple of antibiotics simultaneously. Antibiotics from both

urban and agricultural sources persist in soil thus providing the selective pressure for

emergence of resistance among microorganisms (Thiele-Bruhn, 2003; Segura et al., 2009).

Antibiotics, like Sulphamethoxazole and trimethoprim can be detected in streams also

(Kolpin et al 2002) which may irrigate agricultural lands. Most of antibiotics are produced

by strains of fungi and bacteria that occur naturally in all environments, including soil

(Martin & Liras, 1989). Antibiotic concentrations in some environments have been found

to be high enough to inhibit the growth of many bacterial species. Thus multiple

antimicrobial resistances detected in this study may help these bacteria for their survival in

areas to be decontaminated.

The integron has several unique genetic components that allow them to insert gene

cassettes. Multiple gene cassettes can be inserted into the integron gene sequence to confer

a multiple antimicrobial resistant phenotype to the bacteria (Hall and Collis, 1995; Rowe-

Magnus and Mazel, 1999; Ochman et al., 2000). Persistence and horizontal transfer of

antibiotic resistance genes with the help of integrons may be a tool for the survival of

methyl parathion utilizing bacteria in a competitive microenvironment. This could be a

survival strategy for these bacteria to proliferate ahead of others in the environment, since

most possible antibiosis would not affect them. However it is important to repeatedly

monitor these bacterial strains in the environment for their antimicrobial resistance because

ccxxi

integrons constitute a flexible gene pool that can be exchanged among bacteria of the same

as well as different species, by horizontal gene transfer (Hacker & Carniel, 2001).

The occurrence of integrons in natural ecosystems is important because the

environment apparently serves as a source and a sink for agriculture. The integron gene

sequence is unique in that it acts as a site-specific recombination system capable of

capturing or excising novel genetic elements called gene cassettes coding for a wide range

of antimicrobial resistant determinants (Recchia and Hall, 1995). Integrons are also thought

to play a role in the transfer of pathogenic elements (Mazel et al., 1998). However their

association with any gene components responsible for utilization of methyl parathion as

bacterial substrate is not known yet.

Class 1 integrons are present in natural surface waters and are distributed among a

variety of bacterial genera. Integron sequences have also been isolated previously from

environmental samples like soils (Nield et al., 2001; Michael and Andrew 2010). Studies

suggest that poultry products at the farm, processing, and retail levels harbor antibiotic

resistant organisms and the genetic determinants that could potentially disseminate these

genes. We have previously detected integrons in the fecal samples of buffalo and chicken

in some of the sampling areas of the present study. However any link between these sources

of integrons and resistance genes needs to be established by further studies.

Seven Enterobacteriaceae isolates showing highest methyl parathion degrading

activities with enzyme assay ranging from 410 mU/mL (for Proteus vulgaris IVTMP1) to

675 mU/mL (for Enterobacter Sp. NII-14) were selected for sol-gel immobilization. The

16 S rDNA of identified strains showed highest similarities (91 to 99 %) with Proteus

vulgaris strain DTQ-LP12.1, Proteus sp. KRD11, Enterobacter sp. NII-14,

Enterobacteriaceae bacterium ASC10, Citrobacter sp. SR3, Proteus vulgaris strain

IVTMP1 and Proteus vulgaris. Similar methyl parathion degrading bacterial species

including Citrobacter freundil, Proteus vulgaris and Enterobacter sakazakii has also been

reported previously from agricultural soils in other regions of the world (Ma et al., 2001).

Methyl parathion degrading enzymatic activity of short listed consortium suggested

that the activity of the consortium was higher (982 mU/ml) than those of individual isolates

ccxxii

(which ranged from 410 to 675 mU/ml). Previous studies also documented that the

bacterial consortia are more suitable and effective for mineralization of methyl parathion

as compared to separate (pure) bacterial cultures (Munnecke et al., 1976; Arfman et al.,

1997; Ma et al., 2001; Shimazu et al., 2001; Kumar, 2011). Ma et al. (2001) described the

advantage of methyl parathion degrading consortium which allows some bacterial strain(s)

to carry out initial degradation and other associated bacterial species to utilize the

hydrolytic products (p-nitrophenol and dimethyl thiophosphoric acid) as carbon, nitrogen

and phosphorus source. However, bacteria cultivated in pure cultures can be useful to

determine the degradation capability of each species/strains (Alexander, 1994). Co-

operation between members of consortium may help to establish a single metabolic route

for degradation of methyl parathion. An additional advantage of consortium is that,

enzymes capable of degrading one particular organophosphate may also be useful to

hydrolyze other similar compounds (Munnecke and Hsieh, 1976).

Growth pattern of bacterial strains of the consortium was monitored by using

minimal medium with 0.6 mM methyl parathion as source of carbon and phosphorus. The

growth kinetics of the individual isolates in the presence of methyl parathion revealed

different levels of tolerance to methyl parathion. It reveals differences (p<0.05) of

adaptation to chemical stress in different bacterial species.

Immobilized consortium was stored in sealed tubes at 28 oC (for 4 months), and the

number of viable cells and preservation of methyl parathion hydrolase activity was

analyzed during this period. The sol-gel immobilized consortium was studied for stability,

activity and shelf life. Viability studies showed that consortium survived the sol-gel

immobilization process. However, post immobilization survival rate was different in case

of TEOS and sodium silicate based sol gel matrices. As observed in the previous studies,

the cell survival duration and preservation of metabolic activity depends on the type of

material and technique used in sol-gel process. Our results indicate that the bacterial

consortium cell survival was more uniform in sol-gel when it was supplemented with MSM

and methyl parathion either in TEOS or sodium citrate based matrices as compared to other

storage conditions. Survival rate was better in case of sodium silicate based sol gel stored

with or without MSM and methyl parathion as compared to TEOS based sol gel. Our results

ccxxiii

indicate that provision of nutritional requirements to the sol-gel immobilized cells of

different types may help them for better survival. In addition sodium silicate is able to

support long term survival of the consortium as compared to TEOS based sol-gel matrices.

Methyl parathion hydrolase activity was also monitored simultaneously besides cell

survival in sol-gel. The sol-gel immobilized consortium was able to retain its methyl

parathion hydrolase activity during the whole time of preservation. Water content of the

biocers (biological ceramics) is a limiting factor for the survival and enzymatic activity of

the immobilized bacterial cells due to the cells’ susceptibility to destruction by drying and

shrinkage of the silica network (Alexander, 1994). Therefore; we used wet gels with added

selective marker (methyl parathion) for preservation of methyl parathion hydrolase activity

of the immobilized cells. Methyl parathion hydrolase activity remained intact in non-

encapsulated and in encapsulated cells for 120 days in sodium silicate derived matrix.

In addition, successful GFP expression in E. coli DH5α (transformed with pGFPuv)

during the current study in both types of sol-gel matrices further confirmed the preservation

of biological activity in sol gel immobilized cells. Sol gel immobilization has many

advantages over other methods of microbial preservation. Advantages of cell

immobilization over cultures using suspended cells include greater cellular content in the

support, enhanced cellular viability for weeks or months and greater tolerance to high

levels of concentration of pollutants. These advantages may be more pronounced in case

of preservation and utilization of a consortium of different strains for the common

application. The consortium in this study contained Proteus species which are notorious

for the ability to inhibit growth of unrelated strains (Pearson and Mobley, 2008; Soltman

and Bottcher, 2008). As sol gel immobilization restricts cellular mobility within a defined

space (Fennouh et al., 2000; Premkumar et al., 2002 a, b; Yu et al., 2005) therefore

probably sol-gel immobilization had additional advantage in this case for inhibiting the

mobility of Proteus strains and also possibly decreasing free inhibitory interactions

between different bacterial species in the sol-gel as compared to the suspension.

In contrast to most of previous studies on sol-gel, we used relatively a higher

temperature (28 oC) for evaluation of bacterial survival in sol-gel, different indigenous

bacterial strains in combination instead of a single type of strain, and supplemented the sol-

ccxxiv

gel with a pollutant substrate (methyl parathion) for real field applications. The results

indicate that sol-gel immobilized indigenous methyl parathion degrading bacterial

consortium is capable of surviving at elevated temperature along with preservation of

metabolic function (methyl parathion hydrolase activity). These results would be helpful

to extend the knowledge towards the ultimate goal of using sol-gel immobilized bacterial

strains for bioremediation applications.

8. 5. Conclusions

Our study revealed that the pesticide contaminated soils naturally contain

appreciable diversity of bacteria with the combined potential for metal and antimicrobial

resistance and methyl parathion utilization. Co-tolerance to metals and antimicrobials may

help for their survival in changing environments. Naturally occurring isolates are natural

substitutes for molecularly “designed” microorganisms with the combined activities to

inoculate them into the polluted sites. Metal and antibiotic resistant populations will adapt

faster to natural ecosystem for bioremediation of contaminated soils particularly where

heavy metals and organophosphate pesticides both occur as co-contaminants.

The current study reports on an improved method of methyl parathion utilizing

bacterial consortium immobilization. The cellular viability of the bacterial consortium can

be increased by immobilization, especially in sodium silicate based sol-gel matrix. Sol-gel

immobilization of bacterial consortium may help different bacterial species to increase

their survival duration in co-existence for environmental applications. Moreover, current

study can be very useful to solve the burning environmental issue of pesticides pollution

in an agricultural country like Pakistan and can help the authorities to mitigate the alarming

situation in high pesticides application areas of Pakistan.

ccxxv

Comprehensive conclusions

In our studies we focused on various aspects related to pesticides ranging from

screening of PON1 gene polymorphism in susceptible population to methyl parathion

biosensing assay and characterization of bacterial isolates for methyl parathion

biodegradation.

Population based studies revealed differences in the allele frequencies of PON1

gene polymorphisms and serum paraoxonase and arylesterase activities. PON1 genotypes

frequencies of the current study are similar to what has been reported for Indian, Iranian,

Saudi and Israeli populations which may indicate some common ancestral origins of these

populations. The variations of PON1 genotypes frequencies from other populations,

(especially from Chinese, Korean and Japanese) detected in this study would be due to

genetic drift, geographic drift and ethnic variations. This study provides a basic data of

normal non-pesticide exposed healthy population which will be helpful in PON1 related

disease-association studies in the Pakistani population in future. Based on genotypes, it is

reveled that studied population is more prone to paraoxon related OPs as compared to other

pesticides. Pesticide users reporting toxic effects related to OPs/ pesticides are carriers of

less protective alleles and genotypes alongwith lower paraoxonase and arylesterase

activities.

Our study on PON1 gene screening in a Hypercholesterolemia family revealed

association of PON1 MM/QQ genotype and decreased serum paraoxonase and arylesterase

activities with hypercholesterolemia. Decreased serum PON1 activity may add the risk of

atherosclerosis in patients who are simultaneously hypercholesterolaemic due to defective

LDLR protein.

The results of our study on glaucoma patients did not indicate association of PON1

genotype with primary glaucoma in Pakistani population. However, significant differences

of serum paraoxonase and arylesterase activities in glaucoma patients and healthy controls

compared with younger population suggests age dependant association of PON1 with

glaucoma. Studies on local (rather than systemic) PON1 protein levels and activities in

eyes especially in trabecular meshwork may reveal local effect of PON1 enzyme on eye

tissues involved in glaucoma.

ccxxvi

Our preliminary proof of principle experiment on biosensing applications of

GFPuv revealed the potential of GFPuv purified protein for methyl parathion biosensing

applications. The equipment needed is relatively very simple (sampling tubes, purified

GFPuv protein, fluorometer and UV resistant glasses) which is less costly, easy to use and

can be carried to remote areas very easily where needed.

Results of our studies on HriGFP indicated that it has a unique structure which is

distinct from other proteins. Our results (monomeric FP with hydrophilic fluorophore and

non-beta barrel structure of molecule, phylogenetic distinction from other FPs) indicate

that HriGFP is a novel green fluorescent protein which provides us opportunity of smaller

fluorescent molecules as compared to most of other previously known FPs. HriGFP may

have potential application as a reporter protein in future biosensing studies and

biotechnology.

In addition to above experiments, we isolated, identified and characterized bacterial

strains (isolated from agricultural soils) for their ability to use methyl parathion as source

of carbon and phosphorus. Our study revealed that the pesticide contaminated soils

naturally contain appreciable diversity of bacteria with the combined potential for metal

and antimicrobial resistance and methyl parathion utilization. Co-tolerance to metals and

antimicrobials may help for their survival in changing environments. Naturally occurring

isolates are natural substitutes for molecularly “designed” microorganisms with the

combined activities to inoculate them into the polluted sites. Metal and antibiotic resistant

populations will adapt faster to natural ecosystem for bioremediation of contaminated soils

particularly where heavy metals and organophosphate pesticides both occur as co-

contaminants.

The current study reports on an improved method of methyl parathion utilizing

bacterial consortium immobilization. The cellular viability of the bacterial consortium can

be increased by immobilization, especially in sodium silicate based sol-gel matrix. Sol-gel

immobilization of bacterial consortium may help different bacterial species to increase

their survival duration in co-existence for environmental applications. Moreover, current

study can be very useful to solve the burning environmental issue of pesticides pollution

ccxxvii

in an agricultural country like Pakistan and can help the authorities to mitigate the alarming

situation in high pesticides application areas of Pakistan.

10.0. References

Abbas, C. A., & Sibirny, A. A. (2011) Genetic control of biosynthesis and transport

of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

Microbiol. Mol. Biol. Rev. 75(2), 321-360.

Abu-Qare, A. W. & Abou-Donia, M. B. (2001). Inhibition and recovery of maternal

and fetal cholinesterase enzyme activity following a single cutaneous dose of methyl

parathion and diazinon, alone and in combination, in pregnant rats. J. Appl. Toxicol. 21,

307-316.

Adatia, F.A. and Damji, K. F. (2005) Chronic open angle glaucoma review for

primary care physicians. Canadian Family Physician. 51(9), 1229-1237.

Adkins, S., K. N. Gan, M. Mody and B. N. La Du (1993). "Molecular basis for the

polymorphic forms of human serum paraoxonase /arylesterase: glutamine or arginine at

position 191, for the respective A or B allozymes." Am J Hum Genet 52 (3): 598-608.

Afzal, S., Ahmad, M., Mubarik, A., Saeed, F., Rafi, Sh., Saleem, N. & Qureshi, A.

H. (2006). Acute organophosphorus poisoning-an experience. Pak. Armed. Forces. Med.

J. 56(2), 150-156.

Agarwal, S., Apple, D. J., Buratto, L., Ali, J. L. (2003). Textbook of opthalmology;

Jaypee Brotherse Medical Publishers, New Delhi, 3rd ed.

Ahad, K., Anwar, T., Ahmad, I., Mohammad, A., Tahir, S., Aziz, S., & Baloch, U.

K. (2000). Determination of insecticide residues in groundwater of Mardan Division,

NWFP, Pakistan: a case study. WATER. SA-PRETORIA. 26(3), 409-412.

Ahad, K., Mohammad, A., Mehboob, F., Sattar, A., & Ahmad, I. (2006). Pesticide

residues in Rawal Lake, Islamabad, Pakistan. Bulletin. Environment. Cont. Toxicol. 76(3),

463-470.

Ahemad, M., & Malik, A. (2012). Bioaccumulation of heavy metals by zinc

resistant bacteria isolated from agricultural soils irrigated with wastewater. Bacteriol. J. 2,

12-21.

ccxxviii

Ahemed, M. & Khan, M. S. (2011). Pesticide interactions with Soil Microflora:

Importance in bioremediation. In: Microbes and Microbial Technology: Agricultural and

Environmental Applications, Springer. New York, pp: 393-413

Ahmad, R., Ahad, K., Iqbal, R. & Muhammad, A. (2002). Acute Poisoning due to

commercial pesticides in Multan. Pak. J. Med. Sci. 18(3), 227 – 231.

Ajmal, M., Ahmed, W., Sadeque, A., Ali, S. H. B., Bokhari, S. H., Ahmed, N., &

Qamar, R. (2010). Identification of a recurrent insertion mutation in the LDLR gene in a

Pakistani family with autosomal dominant hypercholesterolemia.Molecular Biol.

Rep. 37(8), 3869-3875.

Al-Allaf, F. A., Coutelle, C., Waddington, S. N., David, A. L., Harbottle, R., &

Themis, M. (2010). LDLR-Gene therapy for familial hypercholesterolaemia: problems,

progress, and perspectives. Int. Arch. Med. 3, 36.

Alexander, M. (1994). Biodegradation and Bioremediation. 302 pp. San Diego,

Academic Press,

Ali, B., Zhang, Q., Lim, Y. K, Fang, D., Retnam, L., & Lim, S. K. (2003)

Expression of major HDL-associated antioxidant PON1 is gender dependent and regulated

during inflammation. Free. Radi.c Biol. Med. 34(7), 824–829.

Ali, T., Bhalli, J. A., Rana, S. M., & Khan, Q. M. (2008). Cytogenetic damage in female

Pakistani agricultural workers exposed to pesticides. Environment. Mol. Mutagenesis. 49(5), 374-

380.

Allebrandt, K. V., Souza, R. L., & Chautard-Freire-Maia, E. A. (2002). Variability

of the Paraoxonase Gene (PON1) in Euro-and Afro-Brazilians. Toxicol. Applied.

Pharmacol. 180(3), 151-156.

Al-Mailem, D. M., Al-Awadhi, H., Sorkhoh, N. A., Eliyas, M., & Radwan, S. S.

(2011). Mercury resistance and volatilization by oil utilizing haloarchaea under hypersaline

conditions. Extremophiles. 15(1), 39-44.

Al-Mailem, D. M., Sorkhoh, N. A., Marafie, M., Al-Awadhi, H., Eliyas, M., &

Radwan, S. S. (2010). Oil phytoremediation potential of hypersaline coasts of the Arabian

Gulf using rhizosphere technology. Bioresource. Technol. 101(15), 5786-5792.

Alvarez, G. S., Desimone, M. F., & Diaz, L. E. (2007). Immobilization of bacteria

ccxxix

in silica matrices using citric acid in the sol–gel process. Applied. Microbiol.

Biotech. 73(5), 1059-1064.

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. (2002). An

optical marker based on the UV-induced green-to-red photoconversion of a fluorescent

protein. Proc. Natl .Acad. Sci. 99, 12651–12656.

Anne, L.C. (1999) Glaucoma. Lancet. 354, 1803-1810.

Ansari, M. T., Iqbal, Z.., & Ahmad, B. (1997). Organochlorine pesticide residues

in human blood in the population of Multan (Pakistan). Pak. J. Pharmaceutical Sci. 10(1),

19-28.

Arfmann, H., Timmis, K. N., & Wittich, R. (1997). Mineralization of 4-

Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and

Burkholderia sp. Strain JWS. Applied. Environment. Microbiol. 63(9), 3458-3462.

Arthur, L.S. and Jan, J. (2005) Color Atlas of Opthalmology. World Scientific

Publishers, London, 5th ed.

Asmathbanu, I.., Kaliwal, B.B. (1997). Temporal effect of methyl parathion on

ovarian compensatory hypertrophy, follicular dynamics and estrous cycle in hemicastrated

albino rat. J. Basic Clin. Phys. Pharma. 8(4), 237-254.

ATSDR (Agency for Toxic Substances and Disease Registry) (1990).

Toxicological profile for methyl parathione. Puplic Health Service, US Department of

Health and Human Services, Washington, DC. ATSDR. (1997). Methyl parathion health

threat. Hazard. Subst. Publ. Hlth. 7, 4-5.

Aviram, M., Hardak, E., Vaya, J., Mahmood, S., Milo, S., Hoffman, A., &

Rosenblat, M. (2000). Human serum paraoxonases (PON1) Q and R selectively decrease

lipid peroxides in human coronary and carotid atherosclerotic lesions PON1 esterase and

peroxidase-like activities. Circulation. 101(21), 2510-2517.

Aviram, M., Rosenblat, M., Billecke, S., Erogul, J., Sorenson, R., Bisgaier, C. L., Newton,

R. S., & La, Du. B. (1999). Human serum paraoxonase (PON1) is inactivated by oxidized low

density lipoprotein and preserved by antioxidants. Free. Radical. Biol. Medicine.26(7), 892-904.

Aviram, M., Rosenblat, M., Bisgaier, C. L., Newton, R. S., Primo-Parmo, S. L., &

La Du, B. N. (1998). Paraoxonase inhibits high-density lipoprotein oxidation and

ccxxx

preserves its functions: a possible peroxidative role for paraoxonase. J. Clinical.

Investigation. 101(8), 1581–1590.

Aydin, O, Yacinkaya, S., Eren, E., Ergin, M., Eroglu, M., & Yilmaz, N. (2013).

Diminished arylesterase enzyme activity and total thiol levels in bladder cancer

patients. Clinical. Lab. 59(11-12), 1231-1237.

Aynacioglu, A. S., Cascorbi, I., Mrozikiewicz, P. M., Nacak, M., Tapanyigit, E. E.,

& Roots, I. (1999). Paraoxonase 1 mutations in a Turkish population.Toxicol. Applied.

Pharmacol. 157(3), 174-177.

Ayub, A., Mackness, M. I., Arrol, S., Mackness, B., Patel, J., & Durrington, P. N. (1999).

Serum paraoxonase after myocardial infarction. Arteriosclerosis. Thrombosis. Vascular.

Biology. 19(2), 330-335.

Bafana, A., Krishnamurthi, K., Patil, M., & Chakrabarti, T. (2010). Heavy metal

resistance in Arthrobacter ramosus strain G2 isolated from mercuric salt-contaminated

soil. J. hazardous. Materials. 177(1), 481-486.

Bahrehmand, F., Vaisi-Raygani, A., Rahimi, Z., Ahmadi, R., Kiani, A., Tavilani,

H., & Pourmotabbed, T. (2014). Synergistic effects of BuChE non-UU phenotype and

paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence

on lipid and lipoprotein metabolism and oxidative stress, preliminary report. Lupus. 23(3),

263-272.

Balestra, G. M. & Misaghi, I. J. (1997). Increasing the efficiency of the plate

counting method for estimating bacterial diversity. J. Microbiol. Meth. 30, 111-117.

Banerjee, S., Datta, S., Chattyopadhyay, D., & Sarkar, P. (2011). Arsenic

accumulating and transforming bacteria isolated from contaminated soil for potential use

in bioremediation. J. Environment. Sci. Health. Part A, 46(14), 1736-1747.

Barr, D. B., Allen, R., Olsson, A. O., Bravo, R., Caltabiano, L. M., Montesano, A.,

Nguyen, J., Udunka, S., Walden, D., Walker, R. D., Weerasekera, G., Whitehead, R. D.,

Schober, S. E. & Needham, L. L. (2005). Concentrations of selective metabolites of

organophosphorus pesticides in the United States population. Environment.

Research. 99(3), 314-326.

ccxxxi

Bartoli, S., Bonora, B., Colacci, A., Niero, A., Grilli, S. (1991). DNA damaging

activity of methyl parathion. Res. Communi. Chem. Pathol. Pharm. 71, 209-218.

Batool, A., Jahan, N., Sun, Y., Hanif, A., & Xue, H. (2014). Genetic association of

IDE, POU2F1, PON1, IL1α and IL1β with type 2 diabetes in Pakistani population. Mol.

Biol. Reports. 41(5), 3063-3069.

Baulcombe, D. C., Chapman, S., & Cruz, S. (1995). Jellyfish green fluorescent

protein as a reporter for virus infections. Plant. J. 7(6), 1045-1053.

Behki, R. & Topp, E. (1993). Metabolism of Carbofuran by Rhodococcus TEI.

Abstract 113. 93rd General Meeting of the American Society for Microbiology.

Berliner, J. A & Heinecke, J. W. (1996). The role of oxidized lipoprotein in

atherogenesis. Free. Radic. Biol. Med. 20, 707–727.

Bernal-Hernández, Y. Y., Medina-Díaz, I. M., Barrón-Vivanco, B. S., de Lourdes

Robledo-Marenco, M., Girón-Pérez, M. I., Pérez-Herrera, N. E., & Rojas-García, A. E.

(2014). Paraoxonase 1 and Its Relationship with Pesticide Biomarkers in Indigenous

Mexican Farmworkers. J Occupt. Environment. Medicine. 56(3), 281-290.

Bhalli, J. A., Ali, T., Asi, M. R., Khalid, Z. M., Ceppi, M., & Khan, Q. M. (2009).

DNA damage in Pakistani agricultural workers exposed to mixture of

pesticides.Environmental and molecular mutagenesis, 50(1), 37-45.

Bhalli, J. A., Khan, Q. M., Haq, M. A., Khalid, A. M., & Nasim, A. (2006).

Cytogenetic analysis of Pakistani individuals occupationally exposed to pesticides in a

pesticide production industry. Mutagenesis. 21(2), 143-148.

Bianchini, A., & Monserrat, J. M. (2007). Effects of methyl parathion on

Chasmagnathus granulatus hepatopancreas: Protective role of Sesamol. Ecotoxicol.

Environ. Saf. 67(1), 100-108.

Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., & La Du,

B. N. (2000). Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones

and cyclic carbonate esters. Drug. Metabol. Disposition. 28(11), 1335-1342.

Birjmohun, R. S., Vergeer, M., Stroes, E. S., Sandhu, M. S., Ricketts, S. L., Tanck,

M. W., ... & Boekholdt, S. M. (2009). Both paraoxonase-1 genotype and activity do not

predict the risk of future coronary artery disease; the EPIC-Norfolk Prospective Population

Study. PLoS. One. 4(8), 6809.

ccxxxii

Blahna, M. T., Zalewski, C. A., Reuer, J., Kahlmeter, G., Foxman, B., & Marrs, C.

F. (2006). The role of horizontal gene transfer in the spread of trimethoprim–

sulfamethoxazole resistance among uropathogenic Escherichia coli in Europe and

Canada. J. Antimicrob. Chemo. 57(4), 666-672.

Blasiak, J., Kowalik, J. (1999). Effect of paraoxon-methyl and parathionmethyl on

DNA in human lymphocytes and protective action of vitamin C. Pesticide. Sci. 55, 1182-

186.

Blatter, M. C., James, R. W., Messmer, S., Barja, F. & Pometta, D. (1993).

Identification of a distinct human high-density lipoprotein subspecies defined by a

lipoprotein-associated protein, K-45. Identity of K-45 with paraoxonase. Europ. J.

Biochemist. 211(3), 871-879.

Bokhari, H., Smith, C., Veerendra, K., Sivaraman, J., Sikaroodi, M., & Gillevet, P.

(2010). Novel fluorescent protein from Hydnophora rigida possess cyano emission.

Biochemical and Biophysical Res. Communications. 396(3), 631-636

Borowczyk, K., Tisończyk, J., & Jakubowski, H. (2012). Metabolism and

neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin

hydrolase. Amino. acids. 43(3), 1339-1348.

Bourne, R. R. A. (2012) The optic nerve head in glaucoma. Community. Eye.

Health. J. 25(79-80), 55-57

Brophy, V. H., Jampsa, R. L., Clendenning, J. B., McKinstry, L. A., Jarvik, G. P.,

& Furlong, C. E. (2001). Effects of 5’ regulatory-region polymorphisms on paraoxonase-

gene (PON1) expression. American. J. Human. Genetics. 68(6), 1428-1436.

Brophy, V. H., Jarvik, G. P., Richter, R. J., Rozek, L. S., Schellenberg, G. D., &

Furlong, C. E. (2000). Analysis of paraoxonase (PON1) L55M status requires both

genotype and phenotype. Pharmacogenetics. Genomics. 10(5), 453-460.

Bryk, B., BenMoyal-Segal, L., Podoly, E., Livnah, O., Eisenkraft, A. & Luria, S.

(2005) Inherited and acquired interactions between ACHE and PON1 polymorphisms

modulate plasma acetylcholineseterase and paraoxonase activities. J.Neurochemist. 92,

1216-1227.

Buckley, N. A., Roberts, D., & Eddleston, M. (2004). Overcoming apathy in

research on organophosphate poisoning. BMJ. 329(7476), 1231-1233.

ccxxxiii

Bulbuller, N., Eren, E., Ellidag, H. Y., Oner, O. Z., Sezer, C., Aydin, O., & Yılmaz,

N. (2013). Diagnostic value of thiols, paraoxonase 1, arylesterase and oxidative balance in

colorectal cancer in human. Neoplasma. 60(4), 419-424.

Bulut, M., Selek, S., Bez, Y., Karababa, I. F., Kaya, M. C., Gunes, M., & Aksoy,

N. (2013). Reduced PON1 enzymatic activity and increased lipid hydroperoxide levels that

point out oxidative stress in generalized anxiety disorder. J. Affective. Disorders. 150(3),

829-833.

Buyukkomurcu, F. (2006). Ratların testisleri üzerine metil parathionun etkisi ve

vitamin C ve E’nin koruyucu rolü. Yüksek Lisans Tezi, Gazi Üniversitesi, Ankara. pp. 1-

37

Cabello, G., Valenzuela, M., Vilaxa, A., Durán, V., Rudolph, I., Hrepic, N., et al.

(2001). A rat mammary tumor model induced by the organophosphorus pesticides

parathion and malathion, possibly through acetylcholinesterase inhibition. Environment.

Health. Perspectives. 109(5), 471–479.

Cabral, L., Giovanella, P., Gianello, C., Bento, F. M., Andreazza, R., & Camargo,

F. A. O. (2013). Isolation and characterization of bacteria from mercury contaminated sites

in Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability of a

Pseudomonas putida V1 strain. Biodegradation. 24(3), 319-331.

Cai, L., Liu, G., Rensing, C., & Wang, G. (2009). Genes involved in arsenic

transformation and resistance associated with different levels of arsenic-contaminated

soils. BMC. Microbiol. 9(1), 9-4.

Carter et al., 2004. Cloning of anthozoan fluorescent protein genes. Comparative.

Biochemist. Physiology. Part C. 138, 259-270.

Casida, J. E., & Quistad, G. B. (2004). Organophosphate toxicology: safety aspects

of nonacetylcholinesterase secondary targets. Chemical Res. Toxicol. 17(8), 983-998.

Catano, H., Cueva, J., Cardenas, A., Izaguirre,V., Zavaleta,A., Carranza, E., &

Hernández, A. (2006). Distribution of paraoxonase-1 gene polymorphisms and enzime

activity in a Peruvian population. Environmetal. Molecul. Mutagenesis. 47, 699-706.

ccxxxiv

Catano, H., E. Carranza, C. Huamaní and A. Hernández (2008). "Plasma

Cholinesterase Levels and Health Symptoms in Peruvian Farm Workers Exposed to

Organophosphate Pesticid es." Archives of Environmental Contamination and Toxicology

55 (1): 153-159.

Cattoir, V., Poirel, L., Rotimi, V., Soussy, C. J., & Nordmann, P. (2007). Multiplex

PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing

enterobacterial isolates. J. Antimicrobial. Chemotherapy. 60(2), 394-397.

Chaiken, I. M. and Smith, E. L. (1969). Reaction of a specific tyrosine residue of

papain with diisopropylfluorophosphate. J. Biol. Chem. 244, 4247–4250.

Chalfie, M. (1995). Green Fluorescent Protein. Photochem. Photobiol. 62, 651–

656.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green

fluorescent protein as a marker for gene expression. Science. 263, 802–805.

Chang, I. L., Elner, G., Yue, Y. J., Cornicelli, A., Kawa, J. E., Elner V. M. (1991)

Expression of modified low-density lipoprotein receptors by trabecular meshwork cells.

Curr. Eye. Res. 10(12), 1101-1112.

Chaudhry, G. R. & Chapalamadugu, S. (1991). Biodegradation of halogenated

organic compounds. Microbiol. Rev. 55, 59-79.

Chaudhry, G. R., Ali, A. N., & Wheeler, W. B. (1988). Isolation of a methyl

parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene

from a Flavobacterium sp. Applied and Environment. Microbiol. 54(2), 288-293.

Chaudhry, G. R., Mateen, A., Kaskar, B., Bloda, M., & Riazuddin, S. (2002).

Purification and biochemical characterization of the carbamate hydrolase from

Pseudomonas sp. 50432. Biotechnol. Applied. Biochemist. 36(1), 63-70.

Chen, J., Kumar, M., Chan, W., Berkowitz, G., & Wetmur, J. G. (2003). Increased

influence of genetic variation on PON1 activity in neonates. Environ. Health Perspect.

111, 1403–1409.

Cheng, H. R., & Jiang, N. (2006). Extremely rapid extraction of DNA from bacteria

and yeasts. Biotechnol. Letters. 28(1), 55-59.

http://www.ncbi.nlm.nih.gov/pubmed?term=Elner%20G%5BAuthor%5D&cauthor=true&cauthor_uid=1802612

http://www.ncbi.nlm.nih.gov/pubmed?term=Yue%20YJ%5BAuthor%5D&cauthor=true&cauthor_uid=1802612

http://www.ncbi.nlm.nih.gov/pubmed?term=Cornicelli%20A%5BAuthor%5D&cauthor=true&cauthor_uid=1802612

http://www.ncbi.nlm.nih.gov/pubmed?term=Kawa%20JE%5BAuthor%5D&cauthor=true&cauthor_uid=1802612

http://www.ncbi.nlm.nih.gov/pubmed?term=Elner%20VM%5BAuthor%5D&cauthor=true&cauthor_uid=1802612

ccxxxv

Cherry, N., Mackness, M., Durrington, P., Povery, A., Dippnall, M., Smith, T., &

Mackness, B. (2002). Paraoxonase (PON1) polymorphisms in farmers attributing ill health

to sheep dip. Lancet. 359(9308), 763-764.

Choi, M. K., Kim, K. D., Ahn, K. M., Shin, D. H., Hwang, J. H., Seong, C. N., &

Ka, J. O. (2009). Genetic and phenotypic diversity of parathion-degrading bacteria isolated

from rice paddy soils. J. Microbiol Biotechnol. 19(12), 1679-1687.

Chowdhury, A., Pradhan, S., Saha, M., & Sanyal, N. (2008). Impact of pesticides

on soil microbiological parameters and possible bioremediation strategies. Indian. J.

Microbiol. 48(1), 114-127.

Christie, J. M., Hitomi, K., Arvai, A. S., Hartfield, K. A., Mettlen, M., Pratt, A. J.,

Tainer J. A., & Getzoff, E. D. (2012). Structural tuning of the fluorescent protein iLOV for

improved photostability. J. Biol. Chemist. 287(26), 22295-22304.

Chudakov, D. M., Belousov, V. V., Zaraisky, A. G., Novoselov, V. V., Staroverov,

D. B., Zorov, D. B., Lukyanov, S., Lukyanov, K. A. (2003). Kindling fluorescent proteins

for precise in vivo photolabeling. Nat. Biotechnol. 21(2), 191-194.

Chudakov, D. M., Verkhusha, V.V., Staroverov, D. B., Souslova, E. A., Lukyanov,

S., Lukyanov, K.A. (2004) Photoswitchable cyan fluorescent protein for protein tracking.

Nat. Biotechnol. 22, 1435–1439.

Clinical and Laboratory Standards Institute (CLSI): (2006). Performance Standards

for Antimicrobial Susceptibility Testing; 16th Informational Supplement M100-S16,

Clinical and Laboratory Standards Institute,Wayne

Cohen, C. S. and Allingham, R. R. (2004). The dawn of genetic testing of

glaucoma. Curr. Opin. Ophthalmol. 15(2), 75-79.

Cole, T. B., Jampsa, R. L., Walter, B. J., Arndt, T. L., Richter, R. J, Shih, D. M, et

al. (2003) Expression of human paraoxonase (PON1) during development.

Pharmacogenetics. (13), 1–8.

Cole, T. B., Walter, B. J., Shih, D. M., Tward, A. D., Lusis, A. J., Timchalk, C., &

Furlong, C. E. (2005). Toxicity of chlorpyrifos and chlorpyrifos oxon in a transgenic mouse

model of the human paraoxonase (PON1) Q192R polymorphism. Pharmacogenetics.

Genomics. 15(8), 589-598.

Cormack, B. P., Valdivia, R. H., Falkow, S., (1996). FACS-optimized mutants of

ccxxxvi

the green fluorescent protein (GFP). Gene. 173, 33–38.

Costa, L. G., Cole, T. B., & Furlong, C. E. (2005). Paraoxonase (PON1): from

toxicology to cardiovascular medicine. Acta. Biomed. 76(2), 50-57.

Costa, L. G., Cole, T. B., Jarvik, G. P., & Furlong, C. E. (2003). Functional

genomics of the paraoxonase (PON1) polymorphisms: effects on pesticide sensitivity,

cardiovascular disease, and drug metabolism. Ann. Review. Medicine. 54(1), 371-392.

Cui, Z. L., Zhang, X. Z., Zhang, Z. H., & Li, S. P. (2004). Construction and

application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the

reporter. Biotechnol. Letters. 26(14), 1115-1118.

Cullings, H. M. (2014). Impact on the Japanese Atomic Bomb Survivors of

Radiation Received From the Bombs. Health. Physics. 106(2), 281-293.

Da Silva, H.C., Medina, H.S.G., Fanta, E., Bacila, M. (1993). Sub-lethal effects of

the organophosphate folidol 600 (methyl parathion) on Callichthys callichthys (Pisces:

Teleostei). Comparative Biochem. Phys. Part C: Comparative Pharm. 105(2), 197-201.

Dalkiliç, S. (2006). Metil paration ile muamele edilen sıçan dokularında sülfidril

grubu konsantrasyonu de_i_imlerinin tespit edilmesi. Yüksek Lisans Tezi, Mustafa Kemal

Üniversitesi, Hatay. pp.1-31.

Dancil, K.P.S., Greiner, D.P., Sailor, M.J. (1999) Development of a porous silicon

based biosensor. Mater. Res. Soc. Symp. Proc. 536, 557–62

Daniel, V., Huber, W., & Baver, K. (2002). Association of dichlorodiphenyl

trichloroethne (DDT) 4.4. and DDE 4.4 blood levels with plasma IL.4 Arch. Environment.

Health. 57(6), 541–547.

Davies, H. G., Richter, R. J., Keifer, M., Broomfield, C. A., Sowalla, J., & Furlong,

C. E. (1996). The effect of the human serum paraoxonase polymorphism is reversed with

diazoxon, soman and sarin. Nat. Genetics. 14(3), 334-336.

Davis, K. A., Crow, J. A., Chambers, H. W., Meek, E. C., & Chambers, J. E. (2009).

Racial differences in paraoxonase-1 (PON1): a factor in the health of southerners. Environ.

Health. Perspect. 117(8), 1226-1231.

de Aguiar, L. H., Moraes, G., Avilez, I. M., Altran, A. E., & Corrêa, C. F.

(2004). Metabolical effects of Folidol 600 on the neotropical freshwater fish matrinxa

ccxxxvii

Brycon cephalus. Environment. Research. 95(2), 224-230.

De, Hertogh, G., Aerssens, J., de, Hoogt, R., Peeters, P., Verhasselt, P., Van, Eyken,

P., Ectors. N., Vermeire, S., Rutgeerts, P., Coulie, B., Geboes, K. (2006) Validation of 16S

rDNA sequencing in microbdissected bowel biopsies from Crohn’s disease patients to

assess bacterial flora diversity. J. Pathol. 209(4), 532-39

Deakin, S., Leviev, I., Guernier, S., James, R. W. (2003) Simvastatin modulates

expression of the PON1 gene and increases serumparaoxonase: a role for sterol regulatory

element-binding protein-2. Arterioscler. Thromb. Vasc. Biol. 1; 23(11), 2083-2089.

deGoma, E. M, deGoma, R. L., Rader, D. J. (2008) Beyond high-

density lipoprotein cholesterol levels evaluating high density lipoprotein function

as influenced by novel therapeutic approaches. J. Am. Coll. Cardiol. 51(23), 2199-211.

Delen, N., Durmu_o_lu, E., Güncan, A., Güngör, N., Turgut, C., Burçak, A. (2005).

Türkiye’de Pestisit Kullanımı, Kalıntı Ve Organizmalarda Duyarlilik Azali Sorunlari.

TMMOB. Ziraat Mühendisleri Odası. Türkiye Ziraat Mühendislii. Teknik. Kongresi. Cilt.

2, 629-648.

Desimone, M. F., De Marzi, M. C., Copello, G. J., Fernández, M. M., Malchiodi,

E. L., & Diaz, L. E. (2005). Efficient preservation in a silicon oxide matrix of Escherichia

coli, producer of recombinant proteins. Applied Microbiol. Biotech. 68(6), 747-752.

Dikshith, T. S. S., Raizada, R. B., Singh, V., Pandey, M., Srivastava, M. K. (1991).

Repeated dermal toxicity of tech. HCH and methyl parathion (50 EC) to female rats (Rattus

norvegicus). Indian. J. Exper. Biol. 29, 149-155.

Distelhorst, J. S., & Hughes, G. M. (2003). Open-angle glaucoma. American.

Family. Physician. 67(9), 1937-1944.

Dong, Y. J., Bartlam,M., Sun,L., Zhou, Y. F., Zhang, Z. P., Zhang, C. G., Rao Z.

H. et al. (2005). Crystal structure of methyl parathion hydrolase from Pseudomonas sp

WBC-3. J. Mol. Biol. 353, 655–663.

Draganov, D. I., La Du, B.N., (2004). Pharmacogenetics of paraoxonases: a brief

review. Naunyn-Schmiedebergs Arch. Pharmacol. 369, 78–88.

Drepper T, Huber R, Heck A, Circolone F, Hillmer AK, Buchs J, Jaeger KE (2010)

Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent

ccxxxviii

protein-like proteins as quantitative in vivo real-time reporters. Appl. Environ. Microbiol.

76(17): 5990-5994.

Dumas, D.P., Durst, H.D., Landis, W.G., Raushel, F.M., Wild, J.R., 1990.

Inactivation of organophosphorus nerve agents by the phosphotriesterase from

Pseudomonas diminuta. Arch. Biochem. Biophys. 227, 155–159.

Durrington, P. N., Mackness, B., Mackness, M. I. (2001). Paraoxonase and

atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21, 473–480.

Dykhuizen, D.E. (1998). Santa Rosalia revisited: why are there so many species of

bacteria? Antonie. Van. Leeuwenhoek. 73, 25-33.

Dzyadevich, S. V., Soldatkin, A. P., Shul’ga, A. A., Strikha, V. I., El’-skaya, A.V.

(1994). Conductometric biosensor for determination of organophosphorus pesticides. J.

Anal. Chem. 49, 874–878.

Eckerson, H. W., Wyte, C. M., & La Du, B. N. (1983). The human serum

paraoxonase/arylesterase polymorphism. American. J. Human. Genetics. 35(6), 1126-

1138.

Ellis, L. B., M., Hershberger, C. D., Bryan, E. M. & Wackett, L. P. (2001). The

University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes.

Nucleic. Acids. Research. 29, 340–343.

Ellison, C. A., Crane, A. L., Bonner, M. R., Knaak, J. B., Browne, R. W., Lein, P.

J., & Olson, J. R. (2012). PON1 status does not influence cholinesterase activity in

Egyptian agricultural workers exposed to chlorpyrifos. Toxicol. Applied.

Pharmacol. 265(3), 308-315.

Emese-Eva, Balint & Judit Petres & Maria Szabo & Csongor-Kalman Orban &

Laszlo Szilagyi & Beata Ábraham (2013). Fluorescence of a Histidine-Modified Enhanced

Green Fluorescent Protein (EGFP) Effectively Quenched by Copper (II) Ions. J. Fluoresc.

23, 273–281

Enders, P., Geldmancher-von Mallinckrodt, M., 1980. Ethnische Unterschiede in

der Verteilung der Aktivit7t der menschlichen Serum Paraoxonase (EC 3.1.1.2). Tag.

Dtsch. Ges. Rechtsmedizin 59, 24–28.

Eqani, S.A., Malik, R.N., Katsoyiannis, A., Zhang, G., Chakraborty, P.,

Mohammad, A., Jones, K.C. (2012) Distribution and risk assessment of organochlorine

ccxxxix

contaminants in surface water from river Chenab, Pakistan. J Environ Monit. 14, 1645–

1654.

Eren, E., Yilmaz, N., & Aydin, O. (2013). Functionally defective high-density

lipoprotein and paraoxonase: a couple for endothelial dysfunction in

atherosclerosis. Cholesterol, 2013.

Ergen, A., Kılıcoglu, O., Ozger, H., Agachan, B., & Isbir, T. (2011). Paraoxonase

1 192 and 55 polymorphisms in osteosarcoma. Molecular biology reports, 38(6), 4181-

4184.

Eva-Maria, Siepert, Esther, Gartz, Mehmet Kemal Tur, Heinrich Delbrück,Stefan

Barth and Jochen Büchs. (2012,) Short-chain fluorescent tryptophan tags for on-line

detection of functional recombinant proteins BMC Biotechnology. 12-65

Excoffier, L. and Slatkin, M. (1995). Maximum likelihood estimation of molecular

haplotype frequencies in a diploid population. Mol Biol Evol. 12, 921-927.

Fadi Bou-Abdallah, N., Dennis Chasteen, Michael, P., Lesser. (2006). Quenching

of superoxide radicals by green fluorescent protein. Biochimica et Biophysica Acta. 1760,

1690–1695.

Falkowski, P. G. & Godfrey, L. V. (2008). Electrons, life and the evolution of

Earth’s oxygen cycle. Philosophical Transactions of the Royal Society B-Biological

Sciences. 363, 2705–2716.

Fallah, S., Seifi, M., Ghasemi, A., Firoozrai, M., & Samadikuchaksaraei, A. (2010).

Matrix metalloproteinase‐ 9 and paraoxonase 1 Q/R192 gene polymorphisms and the risk

of coronary artery stenosis in Iranian subjects.Journal of clinical laboratory

analysis, 24(5), 305-310.

Fang, D. H., Fan, C. H., Ji, Q., Qi, B. X., Li, J., & Wang, L. (2012). Differential

effects of paraoxonase 1 (PON1) polymorphisms on cancer risk: evidence from 25

published studies. Molecular biology reports, 39(6), 6801-6809.

Fanta, E., Rios, F.S.A., Romao, S., Vianna, A.C.C., Freiberger, S. (2003).

Histopathology of the fish Corydoras paleatus contaminated with sublethal levels of

organophosphorus in water and food. Ecotoxicol. Environ. Saf. 54, 119-130.

ccxl

Farid, A. S., & Horii, Y. (2012). Modulation of paraoxonases during infectious

diseases and its potential impact on atherosclerosis. Lipids Health Dis, 11, 92.

Fatma, F.V., Ozcan, E., Hakim, C. (2008) Paraoxonase-1 activity in patients with

hyperemesis gravidarum. Redox Report. 13(3), 134-138.

Fennouh, S., Guyon, S., Livage, J., Roux, C. (2000) Sol-Gel Entrapment of

Escherichia coli. J Sol-Gel Sci Technol. 19, 647–649.

Fesenko, D.O., Nasedkina, T.V., Prokopenko, D.V., Mirzabekov, A.D. (2005)

Biosensing and monitoring of cell populations using the hydrogel bacterial microchip.

Biosens Bioelectron. 20(9), 1860-1865

Flammer, J., Haefliger, I. O., Orgul, S., Resink, T. (1999) Vascular dysregulation:

a principal risk factor for glaucomatous damage? J. Glaucoma. 8(3), 212-219

Frommer WB, Davidson MW, Campbell RE. (2009). Genetically encoded

biosensors based on engineered fluorescent proteins. Chem Soc Rev. 38(10), 2833-2841

Fuchsjager-Mayrl, G., Wally, B., Georgopoulos, M., Rainer, G., Kircher, K., Buehl,

W., Amoako-Mensah, T., Eichler, H. G., Vass, C., Schmetterer, L. (2004) Ocular blood

flow and systemic blood pressure in patients with primary openangle glaucoma and ocular

hypertension. Invest Ophthalmol Vis Sci. 45: 834–839

Furlong, C. E., Richter, R. J., Seidel, S. L., & Motulsky, A. G. (1988). Role of

genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the

insecticide metabolites chlorpyrifos oxon and paraoxon. American journal of human

genetics, 43(3), 230-238.

Furlong, C. E., Cole, T. B., Jarvik, G. P., & Costa, L. G. (2002). Pharmacogenomic

considerations of the paraoxonase polymorphisms. Pharmacogenomics, 3(3), 341-348.

Gaita, L., Manzi, B., Sacco, R., Lintas, C., Altieri, L., Lombardi, F., ... & Persico,

A. M. (2010). Decreased serum arylesterase activity in autism spectrum

disorders. Psychiatry research, 180(2), 105-113.

Ganesan, V. (2008). Rhizoremediation of cadmium soil using a cadmium-resistant

plant growth-promoting rhizopseudomonad. Current microbiology,56(4), 403-407.

Gao, J. F., Ellis, L. B. M. and Wackett, L. P. (2010). The University of Minnesota

Biocatalysis/Biodegradation Database: improving public access. Nucleic Acids Research.

ccxli

38, D488–D491.

Garcia, S.J., Abu-Qare, A.W., Meeker-O’Connell, W.A., Borton, A.J., Abou-

Donia, M.B. (2003). Methyl Parathion: A review of health effects. J. Toxicol. Environ.

Health B, 6, 185-210.

García-Martín, E., Martínez, C., Serrador, M., Alonso-Navarro, H., Navacerrada,

F., Agúndez, J. A., & Jiménez-Jiménez, F. J. (2010). Paraoxonase 1 (PON1)

polymorphisms and risk for migraine. Journal of neurology, 257(9), 1482-1485.

Garin, M.C., James, R.W., Dussoix, P., Blanche, H., Passa, P., Froguel, P. (1997)

Paraoxonase polymorphism Met-Leu54 is associated with modified serum concentrations

of the enzyme. A possible link between the paraoxonase gene and increased risk of

cardiovascular disease in diabetes. J. Clin Invest. 99, 62-66.

Geldmacher-von Mallinckrodt, M., & Diepgen, T. L. (1988). The human serum

paraoxonase—polymorphism and specificity. Toxicological & Environmental

Chemistry, 18(2-3), 79-196.

Gilbert, E.S., Walker, AW.., Keasling, J.D. (2003) A constructed microbial

consortium for biodegradation of the organophosphorus insecticide parathion. Appl

Microbiol Biotechnol. 61(1), 77-81

Goldstein, J. L., & Brown, M. S. (2009). The LDL receptor. Arteriosclerosis,

thrombosis, and vascular biology, 29(4), 431-438.

Gong, M., Garige, M., Varatharajalu, R., Marmillot, P., Gottipatti, C., Leckey, L.

C., & Lakshman, R. M. (2009). Quercetin up-regulates paraoxonase 1 gene expression with

concomitant protection against LDL oxidation. Biochemical and biophysical research

communications, 379(4), 1001-1004.

Gonzalez, C. and Bejarano, L.A. (2000). Protein traps: using intracellular

localization for cloning Trends. Cell Biol. 10, 162–165

Gonzalez, V., Huen, K., Venkat, S., Pratt, K., Xiang P., Harley, K.G., Kogut, K.,

Trujillo, C. M., Bradman, A., Eskenazi, B. and Holland, N. T. (2012). Cholinesterase and

paraoxonase (PON1) enzyme activities in Mexican-American mothers and children from

an agricultural community. Journal of Exposure Science and Environmental

Epidemiology. 22(6), 641-648.

ccxlii

Gordon, S. M., Hofmann, S., Askew, D. S., & Davidson, W. S. (2011). High density

lipoprotein: it's not just about lipid transport anymore. Trends in Endocrinology &

Metabolism, 22(1), 9-15.

Govan, J.R., Hughes, J.E., and Vandamme, P. (1996) Bulkhorderia cepacia:

medical taxonomic and ecological issues. J Med Microbiol 45(6), 395-407

Grassi, S., & Netti, R. (2000). Sea water intrusion and mercury pollution of some

coastal aquifers in the province of Grosseto (Southern Tuscany—Italy).Journal of

Hydrology, 237(3), 198-211.

Grdic, M., K., Barisic, L., Rumora, I., Salamunic, M., Tadijanovic, T., Zanic-

Grubisic, R., Psikalova, Z., Fleger-Mestric, D., Juretic, (2008): Genetic Frequencies of

Paraoxonase 1 Gene Polymorphisms in Croatian Population. Croat Chem Acta. 81, 105-

111

Guney, M., Oral, B., Demirin, H., Ozguner, M., Take, G., Mungan, T., Altuntas, I.

(2007). Evaluation of caspase-dependent apoptosis during methyl parathion-induced

endometrial damage in rats: Ameliorating effect of Vitamins E and C. Environ. Toxicol.

Pharm. 23, 221-227.

Gupta, N., Singh, S., Maturu, V. N., Sharma, Y. P., & Gill, K. D. (2011).

Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in

North-West Indian Punjabis. PLoS One, 6(5), e17805.

Gur, M., Aslan, M., Yildiz, A., Demirbag, R., Yilmaz, R., Selek, S. (2006)

Paraoxonase and arylesterase activities in coronary artery disease. Eur J Clin Invest. 36,

779-787.

Gurskaya, N.G., Verkhusha, V.V., Shcheglov, A.S., Staroverov, D.B.,

Chepurnykh, T.V., Fradkov, A.F., Lukyanov, S., Lukyanov, K.A. (2006) Engineering of a

monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat

Biotechnol 24, 461–465

Habi, S. & Daba, H. (2009). Plasmid incidences, antibiotic and metal resistance

among Enterobacteriaceae isolated from Algerian streams. Pak. J. Biol. Sci. 12, 1474-1482

Hacker, J., & Carniel, E. (2001). Ecological fitness, genomic islands and bacterial

pathogenicity. EMBO reports, 2(5), 376-381.

ccxliii

Hall, R. M., and Collis, C. M. (1995). Mobile gene cassettes and integrons: Capture

and spread of genes by site-specific recombination. Mol. Microbiol. 15, 593–600.

Harel, M,, A., Aharoni, L., Gaidukov, B., Brumstein, O., Khersonsky, R., Meged,

H., Dvir, B.G.R., Ravelli, A., Mccarthy, L., Toker, I., Silman, J.L., Sussman, D.S., Tawfik,

(2004): Structure and evolution of the serum paraoxonase family of detoxifying and anti-

atherosclerotic enzymes. Nat Struct Molec Biol. 11(5), 412-419.

Harel, M., Brumshtein, B., Meged, R., Dvir, H., Ravelli, R., McCarthy, A., ... &

Sussman, J. (2007). 3-D structure of serum paraoxonase 1 sheds light on its activity,

stability, solubility and crystallizability. Archives of Industrial Hygiene and Toxicology,

58(3), 347-353.

Harry, W., Eckerson, Collette, M., Wyte, and Bert, N., La Du, (1983) The Human

Serum Paraoxonase/Arylesterase Polymorphism Am J Hum Genet. 35, 1126-1138.

Hazarika, A., Sarkar, S.N., Hajare, S., Kataria, M., Malik, J.K. (2003). Influence of

malathion pretreatment an the toxicity of anilofos in male rats: a biochemical interaction

study. Toxicology 185, 1- 8.

He, F., Chen, S., Tang, X., Gan, Wen, B. (2002). Biological monitoring of

combined exposure to organophosphates and pyrethroids. Toxicol. Lett. 134, 119-124.

Hedrick, C.C., Thorpe, S.R., Fu, M.X., et al.(2000). Glycation impairs high-density

lipoprotein function.Diabetologia. 43, 312-320.

Hegele, R.A. (1999) Genetic prediction of atherosclerosis: Lessons from studies in

native Canadian populations. Clin Chim Acta. 286, 47-61.

Heim, R., Tsien, R.Y. (1996) Engineering green fluorescent protein for improved

brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 6,

178–182

Helms, C.(1990). Salting out Procedure for Human DNA extraction. In The Donis-

Keller Lab - Lab Manual Homepage.

Henry, E., Newby, D. E., Webb, D. J,, Brien, C. (1999) Peripheral endothelial

dysfunction in normal pressure glaucoma. Invest Ophthalmol Vis Sci 40:1710–1714

ccxliv

Hernandez, A. F., O. Lopez, L. Rodrigo, F. Gil, G. Pena, J. L. Serrano, et al. (2005).

"Changes in erythrocyte enzymes in humans long-term exposed to pesticides: influence of

several markers of individual susceptibility." Toxicol Lett 159 (1): 13-21

Hernandez, A. F., Gil, F., Lacasaña, M., Rodríguez-Barranco, M., Gómez-Martin,

A., Lozano, D., & Pla, A. (2013). Modulation of the endogenous antioxidants paraoxonase-

1 and urate by pesticide exposure and genetic variants of xenobiotic-metabolizing

enzymes. Food and Chemical Toxicology,61, 164-170.

Ho, K., Ing, Zhu, H., Rockhold, W.R., Baker, C.R., Kramer, E.R. (2001). Effects

of single or repeated dermal exposure to methyl parathion on behavior and blood

cholinesterase activity in rats. J. Biomed. Sci. 8, 467-474.

Hobbs, H. H., Russell, D. W., Brown, M. S., & Goldstein, J. L. (1990). The LDL

receptor locus in familial hypercholesterolemia: mutational analysis of a membrane

protein. Annual review of genetics, 24(1), 133-170.

Hofer, S.E., Bennetts, B., Chan, A.K., et al. (2006). Association between PON1

polymorphisms, PON activity and diabetes complications. J Diabetes Complic. 20, 322-

328.

Hong, S.H., Song, J., Min, W.K. and Kim, J.Q. (2001) Genetic variations of the

paraoxonase gene in patients with coronary artery disease. Clin Biochem. 34, 475-481.

Horne, I., Harcourt, RL., Sutherland, TD., Russel, RJ., Oakeshott, JG. (2002).

Isolation of a Pseudomonas monteili strain with a novel phosphotroiesterase. FEMS

Microbial Letters. 206, 51-55.

Huang, A., Teplitski, M., Rathinasabapathi, B., & Ma, L. (2010). Characterization

of arsenic-resistant bacteria from the rhizosphere of arsenic hyperaccumulator Pteris

vittata. Canadian journal of microbiology, 56(3), 236-246.

Huen, K., K., Harley, A., Bradman, B., Eskenezi, N., Holland, (2010) Longitudinal

changes in PON1 enzymatic activities in Mexican-American mothers and children with

different genotypes and haplotypes. Toxicol Appl Pharmacol. 244(2), 181-189

Humbert, R., Adler, D.A., Disteche, C.M., Hassett, C., Omiecinski, C.J., Furlong,

C.E. (1993) The molecular basis of the human serum paraoxonase activity polymorphism.

Nat Genet. 3, 73–76.

ccxlv

Ikonen, E. (2006). Mechanisms for cellular cholesterol transport: defects and

human disease. Physiological reviews, 86(4), 1237-1261.

Imlay, J. A. (2003). Pathways of oxidative damage. Annual Review of

Microbiology. 57, 395–418.

Inagaki, M., Nakagawa-Toyama, Y., Nishida, M., Nakatani, K., Nakaoka, H.,

Kawase, M., ... & Yamashita, S. (2012). Effect of probucol on antioxidant properties of

HDL in patients with heterozygous familial hypercholesterolemia.Journal of

atherosclerosis and thrombosis, 19(7), 643-656.

Inagaki, Y., Mashima, Y., Funayama, T., Ohtake, Y., Fuse, N., Yasuda,

N., Fukuchi, T., Murakami, A., Hotta, Y. (2006)

Paraoxonase 1 gene polymorphisms influence clinical features of open-angle glaucoma.

Graefes Arch Clin Exp Ophthalmol. 244(8):984-990.

IPCS (1993). Environmental health criteria No. 145: methyl parathion.

International Programme on Chemical Safety, IPCS/ World Health Organization, Geneva.

Jacob, P., Kaiser, J. C., & Ulanovsky, A. (2014). Ultrasonography survey and

thyroid cancer in the Fukushima Prefecture. Radiation and environmental biophysics,

53(2), 391-401.

James, R.W., Leviev, I., Righetti, A. (2000). Smoking is associated with reduced

serum paraoxonase activity and concentration in patients with coronary artery disease.

Circulation. 101, 2252–2257.

Janssen, P.H., Yates, P.S., Grinton, B.E., Taylor, P.M., Sait, M. (2002). Improved

culturability of soil bacteria and isolation in pure culture of novel members of the divisions

Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ.

Microbiol. 68, 2391-2396.

Jarvik ,G.P., Jampsa, R., Richter, R.J., Carlson, C.S., Rieder, M.J., Nickerson,

D.A.etal. (2003).Novel paraoxonase (PON1) nonsense and missense mutations predicted

by functional genomic assay of PON1 status. Pharmacogenetics. 13(5), 291-295.

Jarvik, G. P., Trevanian, Tsai, N., McKinstry, L. A., Wani, R., Brophy, V. H,

Richter, R. J., et al. (2002) Vitamins C and E intake is associated with increased

paraoxonase activity. Arterioscler Thromb Vasc Biol. 22, 1329–1333.

http://www.ncbi.nlm.nih.gov/pubmed?term=Mashima%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Funayama%20T%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Ohtake%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Fuse%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Yasuda%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Yasuda%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Fukuchi%20T%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Murakami%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=Hotta%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=16411107

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Jarvik%20GP%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Jampsa%20R%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Richter%20RJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Carlson%20CS%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Rieder%20MJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nickerson%20DA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nickerson%20DA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=novel%20paraoxonase%20%28PON1%29%20nonsense%20and%20missense%20mutations%20predicted%20by

ccxlvi

Jarvik, G.P., Rozek, L.S., Brophy, V.H., Hatsukami, T.S., Richter,

R.J., Schellenberg, G.D. et al. (2000) Paraoxonase (PON1) phenotype is

a better predictor of vascular disease than is PON1(192) or PON1(55)

genotype. Arterioscler Thromb Vasc Biol. 20(11), 2441-2447.

Jeong, S.H., Kim, B.Y., Kang, H.G., Ku, H.O., Cho, J.H. (2006). Effect of

chlorpyrifos-methyl on steroid and thyroid hormones in rat F0- and F1-generations.

Toxicology. 220(2-3), 189-202.

Jeyaratnam, J. (1990). Acute pesticide poisoning: a major global health

problem. World Health Stat Q, 43(3), 139-44.

Jiang, Y. J., Deng, Y. J., Liu, X. R., Xie, B. G., & Hu, F. P. (2006). [Isolation and

identification of a bacterial strain JS018 capable of degrading several kinds of

organophosphate pesticides]. Wei sheng wu xue bao= Acta microbiologica Sinica, 46(3),

463-466.

Jokanovic, M., Stukalov, P. V., & Kosanovic, M. (2002). Organophosphate induced

delayed polyneuropathy. Current Drug Targets-Cns & Neurological Disorders, 1(6), 593-

602.

Joshi, S.C,, Mathur, R., Gajraj, A., Sharma, T. (2003). Influence of methyl

parathion on reproductive parameters in male rats. Environ. Toxicol. Pharm. 14, 91-98.

Kahraman, K. (2006). Ratların ince ba_ırsakları üzerine metil parathionun etkisi ve

vitamin C ve E’nin koruyucu rolü.Yüksek Lisans Tezi, Gazi Üniversitesi, 1-52, Ankara.

Kang, J. H., Pasquale, L. R., Rosner, B. A., Willett, W. C., Egan, K. M.,

Faberowski, N., & Hankinson, S. E. (2003a). Prospective study of cigarette smoking and

the risk of primary open-angle glaucoma. Archives of ophthalmology, 121(12), 1762-1768.

Kang, J. H., Pasquale, L. R., Willett, W., Rosner, B., Egan, K. M., Faberowski, N.,

& Hankinson, S. E. (2003b). Antioxidant intake and primary open-angle glaucoma: a

prospective study. American journal of epidemiology, 158(4), 337-346.

Kao, H.T., Sturgis, S., DeSalle, R., Tsai, J., Davis, D., Gruber, D.F., Pieribone V.A.

(2007) Dynamic regulation of fluorescent proteins from a single species of coral. Mar

Biotechnol. 9(6), 733-46.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Jarvik%20GP%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Rozek%20LS%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Brophy%20VH%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hatsukami%20TS%22%5BAuthor%5D



http://www.ncbi.nlm.nih.gov/pubmed?term=%22Schellenberg%20GD%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed/11073850

ccxlvii

Kao, T. H., Chen, Y., Pai, C. H., Chang, M. C., & Wang, A. H. J. (2011). Structure

of a NADPH-dependent blue fluorescent protein revealed the unique role of Gly176 on the

fluorescence enhancement. Journal of structural biology,174(3), 485-493.

Karadas, S., Aslan, M., Gonullu, H., Kati, C., Duran, L., Olmez, S., ... & Demir, H.

(2014). Acetaminophen intoxication is associated with decreased serum paraoxonase and

arylesterase activities and increased lipid hydroperoxide levels. Human & experimental

toxicology, 0960327113511477.

Karanth, S., Pope, C. (2000) Carboxylesterase and A-esterase activity during

maturation and aging: relationship to toxicity of chlorpyrifos and parathion in rats. Toxicol

Sci. 58, 282–289.

Karasawa, S., Araki, T., Nagai, T., Mizuno, H., and Miyawaki, A. (2004). Cyan-

emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence

resonance energy transfer. Biochem. J. 381, 307–312.

Karen, L. (2006) Glaucoma, An insight into disease and therapy. Canadian

Pharmacists Association, Canada, 1st ed.

Karunya, S.K. and Reetha, D. (2012) Efficiency of Bacterial Isolates in the

Degradation of Malathion and Parathion. International Journal of Pharmaceutical &

Biological Archives. 3(3), 659-665.

Kat, C., Karadas, S., Aslan, M., Gonullu, H., Duran, L., & Demir, H. (2014). Serum

Paraoxonase and Arylesterase Activities and Oxidative Stress Levels in Patients with SSRI

Intoxication. The Journal of membrane biology, 247(1), 17-21.

Kathiresan, S., Willer, C. J., Peloso, G. M., Demissie, S., Musunuru, K., Schadt, E.

E., ... & Altshuler, D. (2009). Common variants at 30 loci contribute to polygenic

dyslipidemia. Nature genetics, 41(1), 56-65.

Kelmanson, I. V., and M. V. Matz. 2003. Molecular basis and evolutionary origins

of color diversity in great star coral Montastraea cavernosa (Scleractinia Faviida). Mol.

Biol. Evol. 20, 1125–1133.

Khalid, N., Khalid, H., Nazia, C., Abdul, M., Umbrin, I., Abdul, G., ... &

Muhammad, I. L. (2011) Eco-friendly role of biodegradation against agricultural pesticides

hazards. African Journal of Microbiology Research. 5(3), 177-183.

ccxlviii

Khan, F., Kuprov, I., Craggs, T. D., Hore, P. J., & Jackson, S. E. (2006). 19F NMR

studies of the native and denatured states of green fluorescent protein.Journal of the

American Chemical Society, 128(33), 10729-10737.

Kilic, S. S., Aydin, S., Kilic, N., Erman, F., Aydin, S., & Celik, I. (2005). Serum

arylesterase and paraoxonase activity in patients with chronic hepatitis. World Journal of

gastroenterology, 11(46), 7351.

Kilic, S. S., Aydin, S., Kilic, N., Erman, F., Aydin, S., & Celik, I. (2005). Serum

arylesterase and paraoxonase activity in patients with chronic hepatitis. World Journal of

gastroenterology, 11(46), 7351.

Kim, K. D., Ahn, J. H., Kim, T., Park, S. C., Seong, C. N., Song, H. G., & Ka, J.

O. (2009). Genetic and phenotypic diversity of fenitrothion-degrading bacteria isolated

from soils. Journal of microbiology and biotechnology, 19(2), 113-120.

Kirbas, A., Kirbas, S., Cure, M. C., & Tufekci, A. (2014). Paraoxonase and

arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic

Parkinson’s disease. Journal of Clinical Neuroscience, 21(3), 451-455.

Kishi, M., Hirschhorn, N., Djajadisastra, M., Satterlee, L. N., Strowman, S., &

Dilts, R. (1995). Relationship of pesticide spraying to signs and symptoms in Indonesian

farmers. Scandinavian journal of work, environment & health, 124-133.

Klein-Schwartz, W., & Smith, G. S. (1997). Agricultural and horticultural chemical

poisonings: mortality and morbidity in the United States. Annals of emergency

medicine, 29(2), 232-238.

Koda, Y., Tachida, H., Soejima, M., Takenaka, O. and Kimura, H. (2004)

Population differences in DNA sequence variation and linkage disequilibrium at the PON1

gene. Ann Hum Genet. 68,110-119.

Kokouva, M., Koureas, M., Dardiotis, E., Almpanidou, P., Kalogeraki, A.,

Kyriakou, D., ... & Hadjichristodoulou, C. (2013). Relationship between the paraoxonase

1 (PON1) M55L and Q192R polymorphisms and lymphohaematopoietic cancers in a

Greek agricultural population. Toxicology,307, 12-16.

Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber,

L.B., and Buxton, H.T. (2002). Pharmaceuticals, hormones, and other waste water

ccxlix

contaminants in U.S. streams, 1999-2000: A national reconnaissance. Environmental

Science and Technology. 36, 1202-1211.

Krieg, N.R., and Holt, J.H. (1984) Bergey’s manual of systematic bacteriology.

Vol. 1, Willilam & Welkins, Washington D.C.

Kujiraoka, T., T. Oka, M. Ishihara, T. Egashi ra, T. Fujioka, E. Sait o, et al.

(2000). "A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase

concentration." J Lipid Res 41 (8): 1358-63.

Kumagai, A., Ando, R., Miyatake, H., Greimel, P., Kobayashi, T., Hirabayashi, Y.,

Shimogori, T., & Miyawaki, A. (2013). A bilirubin-inducible fluorescent protein from eel

muscle. Cell, 153(7), 1602-1611.

Kumar, S. (2011) Bioremediation of chlorpyrifos by bacteria isolated from the

cultivated soils. International Journal of Pharma and Bio Sciences 2(3), 359-366

Kumar, A., B. S. Bisht, V. D., Joshi, & T. Dhewa, (2011). Review on

bioremediation of polluted environment: A management tool. Int. J. Environ. Sci. (1),

1079-1093

Kumar, A., Basavaraj, M. G., Gupta, S. K., Qamar, I., Ali, A. M., Bajaj, V., ... &

Dorairaj, S. K. (2007). Role of CYP1B1, MYOC, OPTN and OPTC genes in adult-onset

primary open-angle glaucoma: predominance of CYP1B1 mutations in Indian

patients. Molecular vision, 13, 667-676

Kwong, T.C. (2002). Organophosphates pesticides: biochemistry and clinical

toxicology. Ther Drug Monit. 24, 144-149.

La Du, B.N., Piko, J.I., Eckerson, H.W., Vincent-Viry, M., Siest, G.,(1986). An

improved method for phenotyping individuals for the human serum paraoxonase

arylesterase polymorphism. Ann. Biol.Clin. 44, 369–372.

Labas, Y.A., Gurskaya, N.G., Yanushevich, Y.G., Fradkov, A.F., Lukyanov, K.A.,

Lukyanov, S.A., Matz, M.V. (2002) Diversity and evolution of the green fluorescent

protein family. Proc Natl Acad Sci USA. 99, 4256–4261.

Lazaros, L. A., Xita, N. V., Hatzi, E. G., Kaponis, A. I., Stefos, T. J., Plachouras,

N. I., Makrydimas, G. V., Sofikitis, N. V., Zikopoulos, K. A., Georgiou, I. A. (2011).

ccl

Association of paraoxonase gene polymorphisms with sperm parameters. J

Androl. 32(4):394-401.

Leckey, L.C., Garige, M., Varatharajalu, R., Gong, M., Nagata, T., Spurney, C.F.

et al. (2010) Quercetin and ethanol attenuate the progression of atherosclerotic plaques

with concomitant up regulation of paraoxonase1 (PON1) gene expression

and PON1 activity in LDLR-/- mice. Alcohol Clin Exp Res. 34(9),1535-42.

Lee, B. W., London, L., Paulauskis, J., Myers, J., & Christiani, D. C. (2003).

Association between human paraoxonase gene polymorphism and chronic symptoms in

pesticide-exposed workers. Journal of occupational and environmental medicine, 45(2),

118-122.

Lee, P. C., Rhodes, S. L., Sinsheimer, J. S., Bronstein, J., & Ritz, B. (2013).

Functional paraoxonase 1 variants modify the risk of Parkinson's disease due to

organophosphate exposure. Environment international, 56, 42-47.

Leeman‐Neill, R. J., Brenner, A. V., Little, M. P., Bogdanova, T. I., Hatch, M.,

Zurnadzy, L. Y., ... & Nikiforov, Y. E. (2013). RET/PTC and PAX8/PPARγ chromosomal

rearrangements in post‐Chernobyl thyroid cancer and their association with iodine‐131

radiation dose and other characteristics. Cancer, 119(10), 1792-1799.

Lefkowitz, R.J., Hoffman, B.B., Taylor, P. (1996). Neurotransmission: the

autonomic and somatic motor nervous systems. In: Hardman J.G., Limbird, L.E., eds.

Goodman and Gilman’s the pharmacological basis of therapeutics. New York, NY,

McGraw Hill. pp. 105-139.

Lein, P. J., Bonner, M. R., Farahat, F. M., Olson, J. R., Rohlman, D. S., Fenske, R.

A., ... & Anger, W. K. (2012). Experimental strategy for translational studies of

organophosphorus pesticide neurotoxicity based on real-world occupational exposures to

chlorpyrifos. Neurotoxicology, 33(4), 660-668.

Leus, F.R., Zwart, M., Kastelein, J.J., Voorbij, H.A. (2001) PON2 gene variants are

associated with clinical manifestations of cardiovascular disease in familial

hypercholesterolaemia patients. Atherosclerosis. 154, 641-649.




ccli

Levario-Carrillo, M., Amato, D., Ostrosky-Wegman, P., González-Horta, C.,

Corona, Y., & Sanin, L. H. (2004). Relation between pesticide exposure and intrauterine

growth retardation. Chemosphere, 55(10), 1421-1427.

Leverıdge, Y.R. (1998). Pesticide poisoning in Costa Rica during 1996. Vet. Hum.

Toxicol. 40(1), 42-44.

Leviev, I., James, R.W., (2000). Promoter polymorphisms of human paraoxonase

PON1 gene and serum paraoxonase activities and concentrations. Arterioscler. Thromb.

Vasc. Biol. 20, 516–521.

Li, B., Nachon, F., Froment, M. T., Verdier, L., Debouzy, J. C., Brasme, B., Gillon,

E., Schopfer, L. M., Lockridge, O., and Masson, P. (2008). Binding and hydrolysis of

soman by human serum albumin. Chem. Res. Toxicol. 21, 421–431.

Li. B., Schopfer, L. M., Grigorean, H., Thompson, C.M., Hinrichs, S.H., Masson,

P., Lockridge., O. (2009) Tyrosines of human and mouse transferrin covalently labeled by

organophosphorous agents: A new motif for binding to proteins that have no active site

serine. Toxicological sciences. 107(1), 144-155

Li, B., Schopfer, L. M., Hinrichs, S. H., Masson, P., and Lockridge, O. (2007).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for

organophosphorus toxicants bound to human albumin at Tyr411. Anal. Biochem. 361, 263–

272.

Li, B., Sedlacek, M., Manoharan, I., Boopathy, R., Duysen, E. G., Masson, P., and

Lockridge, O. (2005). Butyrylcholinesterase, paraoxonase, and albumin esterase, but not

carboxylesterase, are present in human plasma. Biochem. Pharmacol. 70, 1673–1684

Li, L., Zhou, S., Jin, L., Zhang, C., & Liu, W. (2010). Enantiomeric separation of

organophosphorus pesticides by high-performance liquid chromatography, gas

chromatography and capillary electrophoresis and their applications to environmental fate

and toxicity assays. Journal of Chromatography B, 878(17), 1264-1276.

Li, W. F., Matthews, C., Disteche, C. M., Costa, L. G., Furlong, C. E. (1997)

Paraoxonase (PON1) gene in mice: sequencing, chromosomal localization and

developmental expression. Pharmacogenetics. 7:137–44.

cclii

Li, W.F., Costa, L.G., Richter, R.J., Hagen, T., Shih, D.M., Tward, A., Lusis, A.J.,

Furlong, C.E., (2000). Catalytic efficiency determines the invivo efficacy of PON1 for

detoxifying organophosphorus compounds. Pharmacogenetics. 10, 767– 779.

Liang, B., Li, Y. H., & Kong, H. (2013). Serum paraoxonase, arylesterase activities

and oxidative status in patients with insomnia. European review for medical and

pharmacological sciences, 17(18), 2517-2522.

Lin, T.J., Jiang, D.D., Hung, D.Z., Yang, D.Y., Hsu, C.L., Tsai, M.S., (2002).

Paraoxonase activities in Minnan and Hakka Kaohsiung. J. Med. Sci. 18, 551– 556.

Little, M. P., Kukush, A. G., Masiuk, S. V., Shklyar, S., Carroll, R. J., Lubin, J. H.,

... & Likhtarev, I. A. (2014). Impact of Uncertainties in Exposure Assessment on Estimates

of Thyroid Cancer Risk among Ukrainian Children and Adolescents Exposed from the

Chernobyl Accident. PloS one, 9(1), e85723.

Litvinov, D., Mahini, H., & Garelnabi, M. (2012). Antioxidant and anti-

inflammatory role of paraoxonase 1: implication in arteriosclerosis diseases.North

American journal of medical sciences, 4(11), 523-532

Liu, P., Song, X., Yuan, W., Wen, W., Wu, X., Li, J., Chen, X. (2006). Effects of

cypermethrin and methyl parathion mixture on hormone levels and immune function in

Wistar rats. Archives Toxicol. 80(7), 449-457.

Livage, J., (1997). Sol gel processes. Current Opinion in Solid State and Materials

Science. (2),132-138.

Li, Y., Agrawal, A., Sakon, J., Beitlea, R. R. (2001) Characterization of metal

affinity of green fluorescent protein and its purification through salt promoted,

immobilized metal affinity chromatography. Journal of Chromatography A. 909, 183–190

Lockridge, O., & Schopfer, L. M. (2010). Review of tyrosine and lysine as new

motifs for organophosphate binding to proteins that have no active site serine. Chemico-

biological interactions, 187(1), 344-348.

ccliii

Loening, A. M., Fenn, T. D., & Gambhir, S. S. (2007). Crystal Structures of the

Luciferase and Green Fluorescent Protein from< i> Renilla reniformis</i>.Journal of

molecular biology, 374(4), 1017-1028.

Luersen, K., Schmelzer, C., Boesch-Saadatmandi, C., Kohl, C., Rimbach, G., &

Döring, F. (2011). Paraoxonase 1 polymorphism Q192R affects the pro-inflammatory

cytokine TNF-alpha in healthy males. BMC research notes, 4(1), 141.

Ma, L.O.H., Michelle, M.B., Gustavo, Y.O., Enrique, S.S. (2001) Biodegradation

of methyl-parathion by bacteria isolated of agricultural soil. Rev Int Contam Ambient 17(3),

147-155

Mackness MI, Arrol S, Durrington PN. (1991) Paraoxonase prevents accumulation

of lipoperoxides in low-density lipoprotein. FEBS Lett. 286, 152-154.

Mackness, B., & Mackness, M. (2012). The antioxidant properties of high-density

lipoproteins in atherosclerosis. Panminerva medica, 54(2), 83-90

Mackness, B., Davies, G.K., Turkie, W., Lee, E., Roberts, D.H., Hill, E., Roberts,

C., Durrington, P.N., Mackness, M.I. (2001) Paraoxonase status in coronary heart disease:

are activity and concentration more important than genotype? Arterioscler Thromb Vasc

Biol. 21, 1451-457.

Mackness, B., Durington, P., McElduff, P., Yarnell, J., Azam, N., Watt. (2003) Low

paraoxonase activity predicts coronary events in the caterphilly prospective study.

Circulation. 107, 2775-2779.

Mackness, B., Durrington, P.N., Boulton, A.J.M., Hine, D., Mackness, M.I. (2002)

Serum paraoxonase activity in patients with type I diabetes compared to healthy controls.

Eur J Clin Invest. 32:259–264.

Mackness, B., Mackness, M. I., Arrol, S., Turkie, W., Julier, K., Abuasha, B., ... &

Durrington, P. N. (1998). Serum paraoxonase (PON1) 55 and 192 polymorphism and

paraoxonase activity and concentration in non-insulin dependent diabetes

mellitus. Atherosclerosis, 139(2), 341-349.

Mackness, B., Mackness, M.(2010). Anti-inflammatory properties of paraoxonase-

1 in atherosclerosis.In: Reddy ST, ed. Paraoxonases in inflammation, infection and

toxicology.Dordrecht: Humana Press, 143-151.

ccliv

Mackness, B., Mackness, M.I., Arrol, S., Turkie, W. and Durrington, P.N. (1998).

Effect of the Human serum paraoxonase 55 and 192 genetic polymorphism on the

protection by high density lipoprotein against low density lipoprotein oxidative

modification. Eur J Biochem. 423, 57-60.

Mackness, M., & Mackness, B. (2013). Targeting paraoxonase-1 in

atherosclerosis. Expert opinion on therapeutic targets, 17(7), 829-837.

Mackness, M.I., Arrol, S., Abbott, C., Durrington, P.N. (1993) Protection of low-

density lipoprotein against oxidative modification by high-density lipoprotein associated

paraoxonase. Atherosclerosis. 104, 129-135.

Mahrooz, A., Gohari, G., Hashemi, M. B., Zargari, M., Musavi, H., Abedini, M.,

& Alizadeh, A. (2012). R-carrying genotypes of serum paraoxonase (PON1) 192

polymorphism and higher activity ratio are related to susceptibility against ischemic

stroke. Molecular biology reports, 39(12), 11177-11185.

Maitra, S.K., Sarkar, R. (1996). Influence of methyl parathion on gametogenic and

acetylcholinesterase activity in the testis of whitethroated munia (Lonchura malabarica)”,

Archives Environ. Contam. Toxicol. 30, 384-389.

Malashkevich VN, Subach OM, Almo SC, Verkhusha VV. Crystal structure of

TagRFP fluorescent protein (PDB ID: 3M22; unpublished data).

Malin, R., Lehtinen, S., Luoma, P., Näyhä, S., Hassi, J., Koivula, T., & Lehtimäki,

T. (2001). Serum lipid levels and M/L55 allele distribution of HDL paraoxonase gene in

Saami and Finnish men. International journal of circumpolar health, 60(1), 16-24.

Marchegiani, F. M., Marra, F., Olivieri, M., Cardelli, R.W., James, M., Boemi, C.,

Franceschi (2008): Paraoxonase 1: genetics and activities during aging. Rejuv Res. 11(1),

113-127.

Marchegiani, F., M., Marra, F., Olivieri, M., Cardelli, R.,W., James, M., Boemi, C.,

Franceschi (2008): Paraoxonase 1: genetics and activities during aging. Rejuv Res. 11(1),

113-127.

Maria Palfi, Erika Kovacs, Laszlo Szilagy, Ildiko Miklossy, Beata Ábrahám,

Szabolcs Lany. (2010). Fluorescence Quenching Analysis Of Histidine tagged Enhanced

Green Fluorescent Protein. U.P.B. Sci. Bull., Series B, 72(2), (2010)

cclv

Maroni, M., & Fait, A. (1993). Health effects in man from long-term exposure to

pesticides. A review of the 1975-1991 literature. Toxicology, 78(1-3), 1-180.

Martin, J. F., & Liras, P. (1989). Organization and expression of genes involved in

the biosynthesis of antibiotics and other secondary metabolites. Annual Reviews in

Microbiology, 43(1), 173-206.

Mastorikou, M., Mackness, M., Mackness, B. (2006). Defective metabolism of

oxidized phospholipidby HDL from people with type 2 diabetes. Diabetes. 55, 3099-103.

Mathew, G., Vijayalaxmi, K.K., Abdul, Rahiman, M. (1992). Methyl

parathioninduced sperm shape abnormalities in Mouse. Mutation Res. 280,169-173.

Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov,

M.L., and Lukyanov, S.A. (1999). Fluorescent proteins from nonbioluminescent anthozoa

species. Nature Biotechnol. 17, 969-973.

Mazel, D., Dychinco, B., Webb, V. A., and Davies, J. (1998). A distinctive class

of integron in the Vibrio cholerae genome. Science. 280, 605–608.

McCauley, L. A., Anger, W. K., Keifer, M., Langley, R., Robson, M. G., &

Rohlman, D. (2006). Studying health outcomes in farmworker populations exposed to

pesticides. Environmental health perspectives, 953-960.

McGwin, G. Jr., McNeal, S., Owsley, C., Girkin, C., Epstein, D., Lee, P. P (2004)

Statins and other cholesterol-lowering medications and the presence of glaucoma. Arch.

Ophthalmol. 122(6), 822-826.

Mckeown-Eyssen, C. Baines., D.E.C. Cole, N. Riley, R.F, Tyndale, l. Marshall, V.

Jazmaji (2004) Case-control study of genotypes in multiple chemical sensitivity: CYP2D6,

NAT1, NAT2, PON1, PON2 and MTHFR. Int. J. Epidem. 33:1–8

Merhi, M., Raynal, H., Cahuzac, E., Vinson, F., Cravedi, J. P., & Gamet-Payrastre,

L. (2007). Occupational exposure to pesticides and risk of hematopoietic cancers: Meta-

analysis of case-control studies. Cancer Causes Control, 18(10), 1209–1226.

Merzlyak, E. M.,Goedhart, J.,Shcherbo, D.,Bulina, M. E.,Shcheglov, A. S.,

Fradkov, A. F.,Gaintzeva, A.,Lukyanov, K. A.,Lukyanov, S.,Gadella, T. W., and

Chudakov, D. M.(2007). Bright monomeric red fluorescent protein with an extended

fluorescence lifetime. Nat. Methods. 4, 555–557.

cclvi

Michael, S., Qamar, R., Akhtar, F., Khan, W. A., Ahmed, A. (2008).

C677T polymorphism in the methylenetetrahydrofolate reductase gene is

associated with primary closed angle glaucoma. Mol Vis. 14, 661-665.

Mike, J., Sampson, Simon Braschi, Gavin Willis and Sian B. (2005) Astley.

Paraoxonase-1 (PON-1) genotype and activity and in vivo oxidized plasma low-density

lipoprotein in Type II diabetes. Clinical Science. 109, 189–197.

Miller, A.C., M., Stewart, K., Brooks, L., She & N., Page (2002): Depleted

uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay. J

Inorg Biochem. 91(1), 246-252

Miller, S.A., Dykes, D.D. and Polesky, H.F. (1998) A simple salting- out procedure

for extracting DNA from human nucleated cells. Nucl Acid Res 16, 1975.

Milochevitch, C., Khalil, A. (2001) Study of the paraoxonase and platelet activating

factor acetyl hydrolase activities with aging. Prostagl Leukot Essent Fatty Acids. 65, 241–

246.

Mohamed Ali, S., Chia, S. E. (2008). Interethnic Variability of Plasma Paraoxonase

(PON1) Activity towards Organophosphates and PON1 Polymorphisms among Asian

Populations — A Short Review. Ind Health 46, 309–317

Mohammad Perwaiz Iqbal, Abrar Hussain Khan, Naseema Mehboobali, saleem

Perwaiz. (2007) Human paraoxonase and HDL-cholesterol in Pakistani patients with acute

myocardial infarction and healthy adults. Iqbal. Pak J med Sci. 23(5), 659-654.

Moris, P., Alexandre, I., Roger, M., Remacle, J., (1995). Chemiluminescence assay

of organophosphorus and carbamate pesticides. Anal. Chim. Acta 302, 53–59.

Morrison, J.C. and Pollack, I.P. (2003). Glaucoma Science and Practice. Thiemes

medical Publishers, Hongkong, 1st ed.

Moser, V. C., Chanda, S. M., Mortensen, S. R., Padilla, S. (1998) Age -and gender

related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of

esterase activities. Toxicol Sci. 46, 211–22.

Motti, C., Dessi, M., Gnasso, A., Irace, C., Indigeno, P., Angelucci, C.B.,

Bernardini, S., Fucci, G., Federici, G. and Cortese, C. (2001). A multiplex PCR-based

DNA assay for the detection of paraoxonase gene cluster polymorphisms. Atherosclerosis.

158, 35-40.

cclvii

Mozaffarieh, M., Grieshaber, M.C. and Flammer, J. (2008). Oxygen and blood

flow: players in the pathogenesis of glaucoma. Molecular Vision. 14, 224-233

Mueller, R. F., Hornung, S., Furlong, C. E., Anderson, J., Giblett, E. R, Motulsky,

A. G. (1983) Plasma paraoxonase polymorphism: a new enzyme assay, population, family,

biochemical and linkage studies. Am J Hum Genet. 35, 393–408.

Mukherjee, A., Weyant, K. B., Walker, J., & Schroeder, C. M. (2012). Directed

evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein

from Pseudomonas putida. Journal of Biological Engineering. 6-20

Muller-Taubenberger, A., Vos, M.J., Bottger, A., Lasi, M., Lai, F.P., Fischer, M.,

Rottner, K. (2006) Monomeric red fluorescent protein variants used for imaging studies in

different species. Eur J Cell Biol. 85, 1119–1129

Munees Ahemed & Abdul malik (2012). Bioaccumulation of Heavy Metals by Zinc

Resistant Bacteria Isolated from Agricultural Soils Irrigated with Wastewater.

Biotechnology Journal. 2(1), 12-21

Munnecke, D. M., & Hsieh, D. P. (1976). Pathways of microbial metabolism of

parathion. Applied and environmental microbiology, 31(1), 63-69.

Murachi, T., Inagami, T., and Yasui, M. (1965). Evidence for alkylphosphorylation

of tyrosyl residues of stem bromelain by diisopropylphosphorofluoridate. Biochemistry. 4,

2815–2825.

Murphy, H. H., Hoan, N. P., Matteson, P., & Morales Abubakar, A. L. C. (2002).

Farmers' self-surveillance of pesticide poisoning: a 12-month pilot in northern

Vietnam. International journal of occupational and environmental health, 8(3), 201-211.

Murtaza, M., Kisat, M., Daniel, H., & Sonawalla, A. B. (2009). Classification and

clinical features of headache disorders in Pakistan: a retrospective review of clinical

data. PloS one, 4(6), e5827.

Muttray, A., Spelmeyer, U., Degirmenci, M., Jung, D., Backer, G., Hill, G. (2005).

Acute effects of low doses of methyl parathion on human EEG. Environ. Toxicol. Pharm.

19(3), 477-483.

Nadine Nassif, Odile Bouvet, Marie Noelle Rager, Cecile Roux, Thibaud

Coradin & Jacques Livage (2002). Living bacteria in silica gels. Nature Materials. 1, 42 -

44

cclviii

Namba, T., Nolte, C. T., Jackrel, J., & Grob, D. (1971). Poisoning due to

organophosphate insecticides: acute and chronic manifestations. The American journal of

medicine, 50(4), 475-492.

Narayana, K., Prashanthi, N., Nayanatara, A., Kumar, H.H.C., Abhilash, K., Bairy,

K.L. (2005). Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate)

on sperm morphology and sperm count, but not fertility, are associated with decreased

ascorbic acid level in the testis. Mutation Res. 588, 28-34

Nafees, M., Jan, M. R., & Khan, H. (2008). Pesticide Use in Swat Valley, Pakistan:

Exploring Remedial Measures to Mitigate Environmental and Socioeconomic

Impacts. Mountain Research and Development, 28(3), 201-204.

Naz, N., H. K. Young, N. Ahmed & G. M. Gadd (2005). Cadmium accumulation

and DNA homology with metal resistant genes in sulfate reducing bacteria. Applied

Environ. Microbiol. 71, 4610-4618

Neslihan Soran, Ozlem Altindag, hale Cakir, Hakim Celik, Ahmet Demirkot,

Nurten Aksoy. (2008) Assessment of paraoxonase activities in patients with knee

osteoarthritis. Redox Report. 13(5):194-198.

Neufeld, A.H., Sawada, A. and Becker, B. (1999). Inhibition of nitric oxide

synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat

model of chronic glaucoma. Proceedings of the National Academy of Sciences, USA. 96,

9944-9948

Nield, B. S., Holmes, A. J., Gillings, M. R., Recchia, G. D., Mabbutt, B. C.,

Nevalainen, K. M. and Stokes, H. W. (2001). Recovery of new integron classes from

environmental DNA. FEMS Microbiol. Lett. 195, 59–65.

Nies, D. H. (1992). Resistance to cadmium, cobalt, zinc, and nickel in

microbes. Plasmid, 27(1), 17-28.

Ng, C.J., Bourquard, N., Grijalva, V., Hama, S., Shih, D.M., Navab, M., Fogelman,

A.M., Lusis, A.J., Young, S., Reddy, S.T. (2006) Paraoxonase-2 deficiency aggravates

atherosclerosis in mice despite lower apolipoprotein-Bcontaining lipoproteins: anti-

atherogenic role for paraoxonase-2. J Biol Chem. 281, 29491-500.

cclix

Nogueira, C. P., Evans, D. A. P., & La Du, B. N. (1993). The paraoxonase

polymorphism in a Saudi Arabian population. Pharmacogenetics and Genomics, 3(3),

144-149.

Novak, F., Vavrova, L., Kodydkova, J., Novak Sr, F., Hynkova, M., Zak, A., &

Novakova, O. (2010). Decreased paraoxonase activity in critically ill patients with

sepsis. Clinical and experimental medicine, 10(1), 21-25.

Ochman, H., Lawrence, J.G., and Groisman, E.A. (2000). Lateral gene transfer

and the nature of bacterial innovation. Nature. 405, 299–304.

Oliver, J. and Cassidy, L. (2005). Ophthalmology at a glance. Blackwell

Publishing, London 1st ed.

Oshiro, K., Kakuta, T., Sakai, T., Hirota, H., Hoshino, T., Uchiyama, T. (1996).

Biodegradation of Organophosphorus Insecticides by Bacteria Isolated from Turf Green

Soil. J. Ferment. Bioeng. 82, 299-305.

Ozturk, O., Kağnici, Ö. F., Öztürk, T., Durak, H., Tüzüner, B. M., Kisakesen, H. I.,

... & Isbir, T. (2009). 192R allele of paraoxanase 1 (PON1) gene as a new marker for

susceptibility to bladder cancer. Anticancer research,29(10), 4041-4046.

Palleroni, N. (1984). Gram-negative aerobic rods and cocci. In: Bergey’s manual

of Systemic Bacteriology. Williams & Wilkins, Washington D.C., pp. 140-198

Pearson, M.M., Mobley, H.L. (2008) Repression of motility during fimbrial

expression: identification of 14 mrpJ gene paralogues in Proteus mirabilis. Mol Microbiol.

69(2), 548-558.

Pedelacq, J.. D,, Cabantous, S., Tran, T., Terwilliger, T.C., Waldo, G.S. (2005).

Engineering and characterization of a superfolder green fluorescent protein. Nat

Biotechnol. 24,79–88

Peeples, E. S., Schopfer, L. M., Duysen, E. G., Spaulding, R., Voelker, T.,

Thompson, C. M., and Lockridge, O. (2005). Albumin, a new biomarker of

organophosphorus toxicant exposure, identified by mass spectrometry. Toxicol. Sci. 83,

303–312.

Pendic, B., N. Barjaktarovic, V. Kostic (1980): Chromosomal aberrations in

persons accidentally irradiated in Vinca 19 years ago. Radiat Res. 81, 478-482.

cclx

Penna, T. C. V., Ishii, M., de Souza, L. C., & Cholewa, O. (2004). Expression of

green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth

conditions. African Journal of Biotechnology, 3(1), 105-111

Pepi, M., Protano, G., Ruta, M., Nicolardi, V., Bernardini, E., Focardi, S. E., &

Gaggi, C. (2011). Arsenic-resistant Pseudomonas spp. and Bacillus sp. bacterial strains

reducing As (V) to As (III), isolated from Alps soils, Italy. Folia microbiologica, 56(1),

29-35.

Pepi, M., Volterrani, M., Renzi, M., Marvasi, M., Gasperini, S., Franchi, E., &

Focardi, S. E. (2007). Arsenic‐ resistant bacteria isolated from contaminated sediments of

the Orbetello Lagoon, Italy, and their characterization. Journal of applied

microbiology, 103(6), 2299-2308.

Peres, F., Moreire, C. J., Rodrigues, M. K., & Claudio, L. (2006). Risk perception

and communication regarding pesticide use in rural work: A case study in Rio de Janeiro

State, Brazil. International Journal of Occupational and Environmental Health, 12(4),

400–407.

Phuntuwate, W., Suthisisang, C., Koanantakul, B., Mackness, M. I., & Mackness,

B. (2005). Paraoxonase 1 status in the Thai population. Journal of human genetics, 50(6),

293-300.

Pino, N. J., Domínguez, M. C., & Peñuela, G. A. (2011). Isolation of a selected

microbial consortium capable of degrading methyl parathion and p-nitrophenol from a

contaminated soil site. Journal of Environmental Science and Health, Part B, 46(2), 173-

180.

Poh, R., S. Muniandy, (2007) Ethnic variations in paraoxonase 1 polymorphism in

the Malaysian population. Southeas Asian J Trop Med Public Health. 38(2), 392-397.

Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., Cormier, M.J.

(1992). Primary structure of the Aequorea victoria green fluorescent protein. Gene. 111,

229-233

Precourt, L.P., Amre, D., Denis, M.C., Lavoie, J.C., Delvin, E., Seidman, E., Levy,

E. (2011). The three gene paraoxonase family: physiologic roles, actions and regulation.

cclxi

Atherosclerosis, 214, 20-36

Premkumar, J.R., Rosen, R., Belkin, S., Lev, O. (2002,a) Sol-gel luminescence

biosensors: encapsulation of recombinant E. coli reporters in thick silicate films. Anal Chim

Acta. 462, 11-23

Premkumar, J.R., Sagi, E., Rozen, R., Belkin, S., Modestov, A.D., Lev, O. (2002,b)

Fluorescent bacteria encapsulated in sol-gel derived silicate films. Chem Mater. 14(6),

2676-86

Primo-Parmo S.L., R.C., Sorenson, J., Teiber, B.N., La DU (1996): The human

serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.

Genomics. 33, 498-507.

Protano, G., Riccobono, F., & Sabatini, G. (2000). Does salt water intrusion

constitute a mercury contamination risk for coastal fresh water aquifers?.Environmental

Pollution, 110(3), 451-458.

Qiu, X. H., Bai, W. Q., Zhong, Q. Z., Li, M., He, F. Q., & Li, B. T. (2006). Isolation

and characterization of a bacterial strain of the genus Ochrobactrum with methyl parathion

mineralizing activity. Journal of applied microbiology,101(5), 986-994.

Qiu, Y.L., Sekiguchi, Y., Imachi, H., Kamagata, Y., Tseng, I.C., Cheng, S.S.,

Ohashi, A,, Harada, H. (2004). Identification and isolation of anaerobic, syntropic

phthalate isomer degrading microbes from methanogenic sludges treating wastewater from

terepthalate manufacturing. Appl. Environ. Microbiol. 70, 1617-1626.

Quigley, H. A., & Broman, A. T. (2006). The number of people with glaucoma

worldwide in 2010 and 2020. British Journal of Ophthalmology, 90(3), 262-267.

Quigley, H. A., Tielsch, J. M., Katz, J., & Sommer, A. (1996). Rate of progression

in open-angle glaucoma estimated from cross-sectional prevalence of visual field

damage. American journal of ophthalmology, 122(3), 355-363.

Rani, A. & Goel, (2009). Strategies for Crop improvement in contaminated soils

using metal-tolerant bioinoculates. In: microbial strategies for crop improvement, Khan,

M.S., A. Zaidi & J. Musarrat (Eds.). Springer, Berlin, pp: 105-132

Rao, K.S.P., Rao, K.V.R. (1984). Tissue specific alteration of aminotransferases

and total ATPases in the fish (Tilapia mossambica) under methyl parathion impact.

Toxicol. Lett. 20, 53-57.

cclxii

Raymond, M. and Rousset, F. (1995) An exact test for population differentiation.

Evolution. 49, 1280-1283.

Recchia, G. D., & Hall, R. M. (1995). Plasmid evolution by acquisition of mobile

gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a

secondary site in the IncQ plasmid RSF1010. Molecular microbiology, 15(1), 179-187.

Recio, R., Robbins, W. A., Borja-Aburto, V., Moran-Martínez, J., Froines, J.

R., Hernandez, R. M., Cebrian, M. E. (2001).

Organophosphorous pesticide exposure increases the frequency of sperm sex null

aneuploidy. Environ Health Perspect. 109(12):1237-1240.

Rehner, T.A., Kolbo, J.R., Trump, R., Smith, C., Reid, D. (2000). Depression

among victims of South Missisippi’s methyl parathion disaster. Health Social Work 25(1),

33-39.

Richard, S. A., Frank, E. A., & D'Souza, C. J. (2013). Correlation between

cholinesterase and paraoxonase 1 activities: Case series of pesticide poisoning

subjects. BioImpacts: BI, 3(3), 119-122

Richards, P. G., Johnson, M. K., and Ray, D. E. (2000). Identification of

acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds

and a potential target for cognitive enhancing drugs. Mol. Pharmacol. 58, 577–583.

Robyn, J., Russell, Colin Scott, Colin, J., Jackson,* Rinku Pandey, Gunjan Pandey,

Matthew, C., Taylor, Christopher, W., Coppin, Jian-Wei Liu and John, G., Oakeshott.

(2011). The evolution of new enzyme function: lessons from xenobiotic metabolizing

bacteria versus insecticide-resistant insects. CSIRO 4, 225–248

Rojanapongpun, P. (1999) Concepts of glaucoma. Asian Journal of

Ophthalmology. 1, 12-16.

Rojas-Garcia A.E., Sordo, M., Vega, L., Quintanilla-Vega, B., Solis-Heredia, M.,

Ostroskywegman, P. (2009) The role of paraoxonase polymorphisms in the induction of

micronucleus in paraoxon-treated human lymphocytes. Envir Mol Mut. 50(9), 823-829.

Rojas-García, A. E., M.J., Solís-Heredia, B., Piña-Guzmán, L., Vega, L., López-

Carrillo & L. Quintanilla-Vega.( 2005). Genetic polymorphisms and activity of PON1 in a

Mexican population. Toxicology and Applied Pharmacology. 205, 282-289.

Ron, E. (1998). Ionizing radiation and cancer risk: evidence from

cclxiii

epidemiology.Radiation research, 150(5s), S30-S41.

Rosenblat, M., Gaidukov, L., Khersonsky, O., et al. (2006). The catalytic histidine

dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for

PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of

macrophage cholesterol efflux. J Biol Chem. 281, 7657-65.

Rosenblat, M., Volkova, N., Aviram, M.(2011). Injection of paraoxonase 1 (PON1)

to mice stimulates their HDL and macrophage antiatherogenicity. Biofactors. 37(6):462-

467.

Rowe-Magnus, D. A., and Mazel, D. (1999). Resistance gene capture. Curr.

Opinion Microbiol. 2, 483–488.

Rozalski, A., Kwil, I., Torzewska, A., Baranowska, M., Staczek, P. (2007) Proteus

bacilli: features and virulence factors. Postepy Hig Med Dosw. 61, 204-219.

Ruiz, J., H., Blanche, R.W., James, M.C., Garin, C., Vaisse., G., Chapentier, N.,

Cohen, A., Morabia, P., Passa, P., Froguel (1995): Gln-Arg 192 polymorphism of

paraoxonase and coronary artery disease in type 2 diabetes. Lancet. 346, 869–872.

Saeed, M., Perwaiz Iqbal, M., Yousuf, F. A., Perveen, S., Shafiq, M., Sajid, J., &

Frossard, P. M. (2007). Interactions and associations of paraoxonase gene cluster

polymorphisms with myocardial infarction in a Pakistani population.Clinical

genetics, 71(3), 238-244.

Sanchez-Pena, L. C., Reyes, B. E, Lopez-Carrillo, L., Recio, R., Moran-Martinez,

J., Cebrian, M. E., Quintanilla-Vega. B. (2004).

Organophosphorous pesticide exposure alters sperm chromatin structure in Mexicanagric

ultural workers. Toxicol Appl Pharmacol. 196(1):108-13.

Sanghera, D., N. Saha, C.E., Aston, M.I., Kambohi. (1997). Genetic polymorphism

of paraoxonase and the risk of coronary heart disease. Arterioscler Thromb Vasc Biol. 17,

1067–1073.

Sanghera, D., N., Saha, M.I., Kambohi, (1998a) The codon 55 polymorphism in the

paraoxonase1 gene is not associated with the risk of coronary heart disease in Asian Indian

and Chinese. Atherosclerosis. 136, 217-23.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Volkova%20N%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=Reyes%20BE%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

http://www.ncbi.nlm.nih.gov/pubmed?term=Recio%20R%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

http://www.ncbi.nlm.nih.gov/pubmed?term=Mor%C3%A1n-Mart%C3%ADnez%20J%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

http://www.ncbi.nlm.nih.gov/pubmed?term=Mor%C3%A1n-Mart%C3%ADnez%20J%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

http://www.ncbi.nlm.nih.gov/pubmed?term=Cebri%C3%A1n%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

http://www.ncbi.nlm.nih.gov/pubmed?term=Quintanilla-Vega%20B%5BAuthor%5D&cauthor=true&cauthor_uid=15050412

cclxiv

Sanghera, D.K., Saha, N. and Kamboh, I. (1998b) The codon 55 Polymorphism in

the Paraoxonase 1 Gene is not Associated with the Risk of Coronary Heart Disease in Asian

Indians and Chinese. Atherosclerosis. 136, 217-223.

Santoro, M., & Carlomagno, F. (2013). Oncogenic rearrangements driving ionizing

radiation–associated human cancer. The Journal of clinical investigation, 123(11), 4566-

4568.

Santos, N.P.C., A.K.C.R., Santos, S.E.B., Santos, (2005) Frequency of the Q192R

and L55M polymorphisms of the human serum paraoxonase gene (PON1) in ten

Amazonian Ameridian tribes. Genet Mol Biol. 28(1), 36-39.

Sawano, A., Miyawaki, A. (2000) Directed evolution of green fluorescent protein

by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucl

Acids Res 28, E78

Scacchi, R., R.M., Corbo, O., Rickards, G.F., De Stefano, (2003) New data on the

world distribution of paraoxonase (PON1Gln192_Arg) gene frequencies. Hum Biol. 75(3),

365-373.

Schneider, S., Roessli, D. and Excoffier, L. (2000) Arlequin Ver 2.0: A Software

for population genetics data analysis. Genetics and Biometry Lab, Department of

Anthropology, University of Geneva, Switzerland.

Schroder H., A., Heimers, R., Frentzel-Beyme, A., Schott, W., Hoffman (2003):

Chromosome aberration analysis in peripheral lymphocytes of Gulf War and Balkans war

veterans. Radiat Protect Dosim. 103(3), 211-219.

Scott, C., Pandey,G., Hartley, C. J., Jackson,C.J., Cheesman,M.J., Taylor,M.C.,

Pandey,R., et al. (2008). The enzymatic basis for pesticide bioremediation. Indian Journal

of Microbiology. 48, 65–79.

Segura, P. A., François, M., Gagnon, C., & Sauvé, S. (2009). Review of the

occurrence of anti-infectives in contaminated wastewaters and natural and drinking

waters. Environ Health Perspect, 117(5), 675-684.

Seijkens, T., Hoeksema, M. A., Beckers, L., Smeets, E., Meiler, S., Levels, J., ... &

Lutgens, E. (2014). Hypercholesterolemia-induced priming of hematopoietic stem and

progenitor cells aggravates atherosclerosis. The FASEB Journal, 28(5), 2202-2213.

cclxv

Senti, M., Tomas, M., Anglada, R., Elosua, R., Marrugat, J., Covas, M.I., and Fito,

M. (2003). Interrelationship of smoking, paraoxonase activity and leisure time physical

activity: a populationbased study. Eur J Int Med. 14,178–184.

Sepahvand, F., P., Rahimi-Moghaddam, M., Shafiei, S.M.. Ghaffari, M. Rostam-

Shirazi, M., Mahmoudian (2007) Frequency of Paraoxonase 192/55 Polymorphism in an

Iranian Population. J Toxicol Environ Health 70 (13), 1125–1129

Seres, I., Paragh, G., Deschene, E., Fulop, T., Khalil, A. (2004) Study of factors

influencing the decreased HDL associated PON1 activity with aging. Exp Gerontol 39, 59–

66.

Seres, I., Bajnok, L., Harangi, M., Sztanek, F., Koncsos, P., Paragh, G. (2010)

Alteration of PON1 activity in adult and childhood obesity and its relation to adipokine

levels. Adv Exp Med Biol. ,660:129-42.

Serhatlioglu, S., M., Ferit Gursu, F., Gulcu, H., Canata, A., Godekmerdan (2003)

.A levels of paraoxonase and arylesterase activities and malondialdehyde in workers

exposed to ionizing radiation. Cell Biochem Funct. 21, 371-375.

Serrato, M. and Marian, A.J. (1995) A variant of Human paraoxonase/arylesterase

(HUMPONA) gene is a risk factor for coronary heart disease. J Clin Invest. 96, 3005-3008.

Shagin, D.A., et al., (2004). GFP-like proteins as ubiquitous metazoan superfamily:

evolution of functional features and structural complexity. Mol. Biol. Evol. 21, 841–850.

Shaner, N. C., Campbell, R. E., Steinbach, P. A.,Giepmans, B. N., Palmer, A. E.,

and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins

derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572

Sharma, R.P., Reddy, R.V. (1987). Toxic effects of chemicals on the immune

systems. In: Haley, I.J., Berndt, W.O., eds. Handbook of Toxicology. New York, NY:

Hemisphere Publising Corp., pp. 555-591.

Shen, Y. J., Lu, P., Mei, H., Yu, H. J., Hong, Q., & Li, S. P. (2010). Isolation of a

methyl parathion-degrading strain Stenotrophomonas sp. SMSP-1 and cloning of the ophc2

gene. Biodegradation, 21(5), 785-792.

Sherma, J., 1993. Pesticides. Anal. Chem. 65, R40–R54.

Shih, D. M., Gu, L., Xia, Y. R., Navab, M., Li, W. F., Hama, S., ... & Lusis, A. J.

cclxvi

(1998). Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and

atherosclerosis. Nature, 394(6690), 284-287.

Shimazu, M., Mulchandani, A., Chen, W. (2001) Simultaneous degradation of

organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp.

with surface-expressed organophosphorus hydrolase. Biotechnol Bioeng 76(4), 318-324

Shou-zhen, X. (1987). Health effects of pesticides: a review of epidemiologic

research from the perspective of developing nations. American journal of industrial

medicine, 12(3), 269-279.

Siddiqua Jamall, Mohammad Ishaq, Junaid Mahmood Alam, Sana Hussain and

Syed Muhammad Waqar Hussain. (2010) Paraoxonase activity in patients with chronic

renal failure and hepatic insufficiency. Pak. J. Biochem. Mol. Biol. 43(2), 54-57.

Siepert, E. M., Gartz, E., Tur, M. K., Delbrück, H., Barth, S., & Büchs, J. (2012).

Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant

proteins. BMC biotechnology, 12(1), 65.

Silva, D., Cortez, D.M., Cunha-Bastos, J., Louro, S.R. (2004) Methyl parathion

interaction with human and bovine serum albumin. Toxicol Lett. 147(1), 53-61

Singh S, Kumar V, Vashisht K, Singh P, Banerjee BD, Rautela RS, Grover SS,

Rawat DS, Pasha ST, Jain SK, Rai A. (2011). Role of genetic polymorphisms of CYP1A1,

CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers

occupationally exposed to organophosphate pesticides. Toxicol Appl Pharmacol.

257(1):84-92.

Singh, S. K., Tripathi, V. R., Jain, R. K., Vikram, S., & Garg, S. K. (2010). An

antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU 1 and its

thermoalkaline protease. Microbial cell factories, 9, 59-59.

Singh, S., B., Lehmann-Grube, H.W., Goedde, (1984): Cytogenetic effects of

paraoxon and methylparathion on cultured human lymphocytes: SCE, clastogenic activity

and cell cycle delay. Int Arch Environ Health. 54, 192-200.

Sizova, O. I., Kochetkov, V. V., & Boronin, A. M. (2010). Rhizosphere bacteria

Pseudomonas aureofaciens and Pseudomonas chlororaphis oxidizing naphthalene in the

presence of arsenic. Applied biochemistry and microbiology,46(1), 38-43.




cclxvii

Slatkin, M. and Excoffier, L. (1996) Testing for Linkage Disequilibrium in

Genotypic data Using the EM Algorithm. Heredity. 76, 377-383.

Soltmann, U., Bottcher, H. (2008) Utilization of sol-gel ceramics for the

immobilization of living microorganisms. J Sol-Gel Sci Technol. 48, 66-72.

Sorenson, R.C., C.L., Bisgaier, M., Aviram, C., Hsu, S., Billecke, B.N., La Du

(1999): Human serum paraoxonase/arylesterase’s retained hydrophobic N-terminal leader

sequence associates with HDLs by binding phospholipids: apoprotein A-1 stabilizes

activity. Arterioscler Thromb Vasc Biol. 19, 2214-2225.

Sorkhoh, N. A., Al-Awadhi, H., Al-Mailem, D. M., Kansour, M. K., Khanafer, M.,

& Radwan, S. S. (2010a). Agarolytic bacteria with hydrocarbon-utilization potential in

fouling material from the Arabian Gulf coast. International Biodeterioration &

Biodegradation, 64(7), 554-559.

Sorkhoh, N. A., Ali, N., Al-Awadhi, H., Dashti, N., Al-Mailem, D. M., Eliyas, M.,

& Radwan, S. S. (2010b). Phytoremediation of mercury in pristine and crude oil

contaminated soils: Contributions of rhizobacteria and their host plants to mercury

removal. Ecotoxicology and environmental safety, 73(8), 1998-2003.

Sortur, S.M., Kaliwal, B.B. (1999). Effects of methyl parathion formulation on

estrous cycle and reproductive performances in albino rats. Indian J. Exper. Biol. 37(2),

176-178.

Stanley, J. C. (2008). Feedback inhibition of cholesterol synthesis and uptake.Lipid

Technology, 20(1), 17-18.

Steinberg D., S., Parthasarathy, T.E., Carew, J.C., Khoo, J.L., Witztum (1989)

Beyond cholesterol. Modifications of low-density lipoprotein that increase its

atherogenicity. N Engl J Med. 320, 915-924.

Storz, G., and Imlay, J. A. (1999). Oxidative stress. Current Opinion in

Microbiology 2, 188–194.

Strack, R.L., Strongin, D.E., Bhattacharyya, D., Tao, W., Berman, A., Broxmeyer,

H.E., Keenan, R.J., Glick, B.S. (2008) A noncytotoxic DsRed variant for whole-cell

labeling. Nat Methods. 5(11), 955-957.

cclxviii

Strong, P. J. & J. E. Burgess, (2008) treatment method for wine-related ad distillary

wastewater: A review. Bioremediat. J. 12, 70-87

Suleyman, Sirri Kilic, Suleyman Aydin, Nermin Kilic, Fazilet Erman, Suna Aydin,

İlhami Celik. (2005). Serum arylesterase and paraoxonase activity in patients with chronic

hepatitis. World J Gastroenterol. 11(46), 7351-7354.

Sunay, S. Z., Kayaalti, Z., Bayrak, T., & Soylemezoglu, T. (2013). Effect of

paraoxonase 1 192 Q/R polymorphism on paraoxonase and acetylcholinesterase enzyme

activities in Turkish population exposed to organophosphate. Toxicology and industrial

health,

Syed, I.A. and Mughal, K.I (1998) Glaucoma disease pattern in Pakistan. Asian

Journal of Ophthalmology. 1(1), 6

Szanto, A., Harangi, M., Seres, I., Paragh, G., & Zeher, M. (2010). Decreased

human paraoxonase-1 activity in patients with Sjögren's syndrome. International

immunology, 22(7), 605-609.

Tafuri, J., Roberts, J. (1987). Organophosphate poisoning. Ann. Emerg. Med. 16,

193-202.

Tamura, H., Maness, S.C., Dorman, D.C., Gray, L.E., Gaido, K.W. (2001).

Androgen receptor antagonism by the organophosphate insecticide fenitrothion. Toxicol.

Sci. 60, 56-62.

Tao, W., Evans, B. G.,Yao, J.,Cooper, S.,Cornetta, K.,Ballas, C. B.,Hangoc, G.,

and Broxmeyer, H. E.(2007). Enhanced green fluorescent protein is a nearly ideal long-

term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent

protein is not. Stem Cells. 25, 670–678.

Tariq, M. I., Afzal, S., & Hussain, I. (2004). Pesticides in shallow groundwater of

Bahawalnagar, Muzafargarh, DG Khan and Rajan Pur districts of Punjab,

Pakistan. Environment international, 30(4), 471-479.

Tariq, M. I., Afzal, S., Hussain, I., & Sultana, N. (2007). Pesticides exposure in

Pakistan: A review. Environment International, 33(8), 1107-1122.

cclxix

Taylor, H.R. and Keeffe, J.E. (2001) world blindness: a 21st century perspective.

British Journal of Ophthalmology. 85, 261-266

Taylor, P. (1996). Anticholinesterase agents. In: Hardman JG, Limbird LE,

Molinoff PB, Ruddon RW, Gilman AG, eds. Goodman and Gillman’s Pharmacol. Basis

Therapeu. New York, McGraw-Hill. 9, 177-197.

Thiele-Bruhn, S. (2003). Pharmaceutical antibiotic compounds in soils–a

review.Journal of Plant Nutrition and Soil Science, 166(2), 145-167.

Thomas, M., van, H., Mark, R., Jacqueline, de, G., Hattori, H., Kastelein, J.J.,

(2005). Indications that paraoxonase-1 contributes to plasma high density lipoprotein

levels in familial hypercholesterolemia. Journal of lipid research. 46, 445-451.

Thomas, R., Sekhar, G. C., & Parikh, R. (2007). Primary angle closure glaucoma:

a developing world perspective. Clinical & experimental ophthalmology, 35(4), 374-378.

Todd, A., Richmond, Terry, T., Takahashi, Riti Shimkhada, and Jennifer Bernsdorf.

(2000) Engineered Metal Binding Sites on Green Fluorescence Protein. Biochemical and

Biophysical Research Communications. 268, 462–465

Tomas, M., Sentí, M., García-Faria, F., Vila, J., Torrents, A., Covas, M., &

Marrugat, J. (2000). Effect of simvastatin therapy on paraoxonase activity and related

lipoproteins in familial hypercholesterolemic patients. Arteriosclerosis, thrombosis, and

vascular biology, 20(9), 2113-2119.

Torsvik, V., Daae, F.L., Sandaa, R-A., øvreås, L. (1998). Novel techniques for

analyzing microbial diversity in natural and perturbed environments. J. Biotechnol. 64, 53-

62.

Torsvik, V., Goksøyr, J., Daae, F.L. (1990). High diversity in DNA of soil bacteria.

Appl Environ Microbiol. 56, 782-787

Trojanowicz, M., Hitchman, M.L.,(1996). Determination of pesticides using

electrochemical biosensors. Trends Anal. Chem. 15, 38–45.

cclxx

Tsien, R.Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67: 509-

544

Tuovinen, K. (2004). Organophosphate-induced convulsions and prevention of

neuropathological damages. Toxicology. 196, 31-39.

Undeger, U., Institoris, L., Siroki, O., Nehez, M., Desi, I. (2000). Simultaneous

genoand immunotoxicological investigations for early detection of organophosphate

toxicity in rats. Ecotoxicol. Environ. Saf. 45, 43-48.

Uzunhisarcikli, M. (2008). Methyl parathion’un ratlarda hepatotoksik etkisi ve

vitamin C ve vitamin E’nin koruyucu rolü. Doktora Tezi, Gazi Üniversitesi, 1-111, Ankara.

Uzunhisarcikli, M., Kalender, Y., Dirican, K., Kalender, S., Ogutcu, A.,

Buyukkomurcu, F. (2007). Acute, subacute and subchronic administration of methyl

parathion-induced testicular damage in male rats and protective role of vitamins C and E.

Pest. Biochem. Phys. 87, 115-122.

Vaisi-Raygani, A., Ghaneialvar, H., Rahimi, Z., Tavilani, H., Pourmotabbed, T.,

Shakiba, E., ... & Bartels, C. (2011). Paraoxonase Arg 192 allele is an independent risk

factor for three-vessel stenosis of coronary artery disease.Molecular biology reports, 38(8),

5421-5428.

Van, Himbergen, T.M., Roest, M., de, Graaf, J., Jansen, E.H., Hattori, H.,

Kastelein, J.J.(2005). Indications that paraoxonase-1 contributes to plasma high density

lipoprotein levels in familial hypercholesterolemia. Journal of lipid research. 46, 445-451.

Verkhusha, V.V. and Sorkin, A. (2005) Conversion of the monomeric red

fluorescent protein into a photoactivatable probe. Chem Biol. 12, 279–285

Verkhusha, V.V., Lukyanov, K.A., 2004. The molecular properties and

applications of Anthozoa fluorescent proteins and chromoproteins. Nat. Biotechnol. 22,

289–296.

Vural ,N. (2005). Toksikoloji. Ankara Üniversitesi Eczacılık Fakültesi Yayınları.

73, 381.

cclxxi

Wallace, A. J., Mann, J. I., Sutherland, W. H., Williams, S., Chisholm, A., Skeaff,

C. M., ... & Humphries, S. E. (2000). Variants in the cholesterol ester transfer protein and

lipoprotein lipase genes are predictors of plasma cholesterol response to dietary change.

Atherosclerosis, 152(2), 327-336.

Wang, S. and Hazelrigg, T. (1994). Implication for bcd mRNA localization from

spatial distribution of exu protein in Drosophila oogenesis. Nature. 369, 400-403

Wang, X., Fan, Z., Huang, J., Su, S., Yu, Q., Zhao, J., ... & Gu, D. (2003).

Extensive association analysis between polymorphisms of PON gene cluster with

coronary heart disease in Chinese Han population. Arteriosclerosis, thrombosis, and

vascular biology, 23(2), 328-334.

Wen, Y., Jiang, J. D., Deng, H. H., Lan, H., & Li, S. P. (2007). [Effect of mutation

of chemotaxis signal transduction gene cheA in Pseudomonas putida DLL-1 on its

chemotaxis and methyl parathion biodegradation]. Wei sheng wu xue bao= Acta

microbiologica Sinica, 47(3), 471-476.

Wheeler, J.G., Keavney, B.D., Watkins, H., Collins, R., Danesh, J. (2004) Four

paraoxonase gene polymorphisms in 11212 cases of coronary heart disease and 12786

controls: meta-analysis of 43 studies. Lancet. 363, 689-95.

WHO (2001). Organophosphate pesticides in the environment-Integrated Risk

Assessment, Geneva: WHO

Wiedenmann, J., Schenk, A., Rocker, C., Girod, A., Spindler, K. D., and Nienhaus,

G. U.(2002). A far-red fluorescent protein with fast maturation and reduced

oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc. Natl.

Acad. Sci. USA 99, 11646–11651.

Wilmann, P. G., Battad, J., Petersen, J., Wilce, M. C., Dove, S., Devenish, R. J.,

Prescott, M., Rossjohn, J. (2006). The 2.1A crystal structure of copGFP, a representative

member of the copepod clade within the green fluorescent protein superfamily. J. Mol.

Biol. 359(4): 890-900.

cclxxii

Worek, F., Seeger, T., Goldsmith, M., Ashani, Y., Leader, H., Sussman, J. S., ... &

Wille, T. (2014). Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity in vivo

and to detoxify structurally different nerve agents in vitro.Archives of toxicology, 1-10.

Yamada, Y., Ando, F., Niino, N., Miki, T. and Shimokata, H. (2003) Association

of polymorphisms of paraoxonase 1 and 2 genes, alone or in combination, with bone

mineral density in Community- Dwelling Japanese. J Hum Genet 48, 469-475.

Yamamoto, T., Egashıra, T., Yoshida, T., Kuroiwa, Y. (1983). Comparative

metabolism of fenitrothion and methyl parathion in male rats. Acta Pharmacol. Toxicol.

53, 96-102.

Yan, Q. X., Hong, Q., Han, P., Dong, X. J., Shen, Y. J., & Li, S. P. (2007). Isolation

and characterization of a carbofuran‐ degrading strain Novosphingobium sp. FND‐

3. FEMS microbiology letters, 271(2), 207-213.

Yan, y. Goh, Vladimir Frecer, Bow Ho and Jeak, L., Ding. (2002) Rational design

of green fluorescent protein mutants as biosensor for bacterial endotoxin. Protein

engineering. 15(6), 493-502.

Yano, B.L., Young, J.T., Mattsson, J.L. (2000). Lack of carcinogenicity of

chlorpyrifos insecticide in a high-dose, 2-year dietary toxicity study in Fischer 344 rats.

Toxicol. Sci. 53, 135-144.

Yanushevich, Y.G., Staroverov, D.B., Savitsky, A.P., Fradkov, A.F., Gurskaya

N.G., Bulina, M.E., Lukyanov, K.A., Lukyanov, S.A. (2002) A strategy for the generation

of nonaggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511, 11–14

Yasmeen Rahimi, Ann Goulding, Suresh Shrestha, Sweetie Mirpuri, Sapna, K.,

Deo, (2008). Mechanism of copper induced fluorescence quenching of red fluorescent

protein, DsRed. Biochemical and Biophysical Research Communications. 370, 57–61

Yasmeen Rahimi, Suresh Shrestha, Tanushree Banerjee, Sapna, K., Deo. (2007)

Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa.

Analytical Biochemistry. 370, 60–67

cclxxiii

Yildirim, M. (2006). Metil paratiyonun ratlarda nefrotoksik etkisi ve Vitamin Cve

E’nin koruyucu rolü. Yüksek Lisans Tezi, Gazi Üniversitesi, 1-50, Ankara.

Yilmaz, N., Simsek, N., Aydin, O., Yardan, E., Aslan, S., Eren, E., ... & Buyukbas,

S. (2013). Decreased paraoxonase 1, arylesterase enzyme activity, and enhanced oxidative

stress in patients with mitral and aortic valve insufficiency. Clinical laboratory, 59(5-6),

597-604.

You, T., Lv, J., & Zhou, L. (2013). PON1 Q192R and L55M Polymorphisms and

Organophosphate Toxicity Risk: A Meta-Analysis. DNA and cell biology, 32(5), 252-259.

Yu, D., Volponi, J., Chhabra, S., Brinker, C.J., Mulchandani, A., Singh, A.K.

(2005). Aqueous sol-gel encapsulation of genetically engineered Moraxella spp. cells for

the detection of organophosphates. Biosens Bioelectron. 20(7), 1433-37

WHO. Geneva: World Health Organization; 1990. Public Health Impact of

Pesticides Used in Agriculture; p. 88.

W.H.O (1993). Environmental Health Criteria: Methyl parathion. IPCS, Geneva

Wehner, J. M., Murphy-Erdosh, C., Smolen, A., Smolen, T. S. (1987). Genetic

variation in paraoxonase activity and sensitivity to disopropylphosphofluoridate in inbred

mice. Pharmacol Biochem Behav. 28, 317–320.

Weinreb, R. N. and Khaw, P. T. (2004) Primary open-angle glaucoma. Lancet.

363(9422), 1711-1720.

White, P. A., McIver, C. J., & Rawlinson, W. D. (2001). Integrons and Gene

Cassettes in theEnterobacteriaceae. Antimicrobial agents and chemotherapy,45(9), 2658-

2661.

Wiegman, A., de Groot, E., Hutten, B.A., Rodenburg, J., Gort, J., Bakker, H.D.

(2004). Arterial intima-media thickness in children heterozygous for familial

hypercholesterolaemia. Lancet. 363, 369-370.

Wiggs, J. L. (2007). Genetic etiologies of glaucoma. Archives of

ophthalmology,125(1), 30-37.

cclxxiv

Wissinger, B., Tubingen, S.K., Tubingen, U.L., Frankfurt, M.B. and Karger.

(2003). Genetics in ophthalmology. Developments in Ophthalmology. 37, 83-93

Xie, C., Ding, X., Gao, J., Wang, H., Hang, Y., Zhang, H., ... & Miao, L. (2014).

The effects of CES1A2 A (− 816) C and CYP2C19 loss-of-function polymorphisms on

clopidogrel response variability among Chinese patients with coronary heart

disease. Pharmacogenetics and genomics, 24(4), 204-210.

Yen, Y. C., Yang, J. J., Chou, M. C., & Li, S. Y. (2008). Absence of optineurin

(OPTN) gene mutations in Taiwanese patients with juvenile-onset open-angle

glaucoma. Molecular vision, 14, 487-494

Yilmaz, N. (2012). Relationship between paraoxonase and homocysteine:

crossroads of oxidative diseases. Arch Med Sci, 8(1), 138-153.

Yoon, I. H., Chang, J. S., Lee, J. H., & Kim, K. W. (2009). Arsenite oxidation by

Alcaligenes sp. strain RS-19 isolated from arsenic-contaminated mines in the Republic of

Korea. Environmental geochemistry and health, 31(1), 109-117.

Yoshida, N., T. kato, T., Yoshida, K., ogawa, M., Yamashita & Y., Murrooka,

(2002). Bacterium based heavy metal biosorbents: enhanced uptake of cadmium by

Escherichia coli expressing a metallothionein fused to b-galactosidase. Biotechniques, 32,

551-558

Zehra ,H., Amber, I., Ammara, S., Asmat, S., Shamshad, Z. (2009) Expression and

activity of paraoxonase 1 in human catacratous lens tissue. Free radical Biology and

Medicine. 46:1089-1095.

Zhang, R., Cui, Z., Jiang, J., He, J., Gu, X., Li, S. (2005) Diversity of

organophosphorus pesticide-degrading bacteria in a polluted soil and conservation of their

organophosphorus hydrolase genes. Can J Microbiol. 51(4), 337-343

Zhang, X., Sui, H., Li, H., Zheng, J., Wang, F., Li, B., & Zhang, Y. (2014).

Paraoxonase activity and genetic polymorphisms in northern Han Chinese workers

exposed to organophosphate pesticides. Experimental Biology and Medicine, 239(2), 232-

239.

cclxxv

Zhang, Y., Zheng, F., Du, H., Krepinsky, J. C., Segbo, J. A., & Zhou, X. (2006).

Detecting the polymorphisms of paraoxonase (PON) cluster in Chinese Han population

based on a rapid method. Clinica chimica acta, 365(1), 98-103.

Zhang, Z., Hong, Q., Xu, J., Zhang, X., & Li, S. (2006,b). Isolation of fenitrothion-

degrading strain Burkholderia sp. FDS-1 and cloning of mpd gene.Biodegradation, 17(3),

275-283.

Zhu, H., Rockhold, R.W., Baker, R.C., Kramer, R.E., Ho, I.K. (2001). Effects of

single or repeated dermal exposure to methyl parathion on behavior and blood

cholinesterase activity in rats. J. Biomed. Sci. 8, 467-474.

Zolgharnein, H., K., karami, M. M., Assadi, & A. D., Sohrab, (2010). Investigation

of heavy metals biosorption on pseudomonas aeruginosa strain MCCB 102 isolated from

gulf. Asian J. Biotechnol. 2, 99-109

Development of a Biosensor for Methyl Parathion...

Documents

Transcript of Development of a Biosensor for Methyl Parathion...