Development of a Biosensor for Methyl Parathion...
Transcript of Development of a Biosensor for Methyl Parathion...
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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
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COMSATS Institute of Information Technology
Development of a Biosensor for Methyl Parathion
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
CIIT/FA07-PBS-004/ISB
Spring, 2015
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Development of a Biosensor for Methyl Parathion
Detection and Genetic Risk Assessment of Exposed
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
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Final Approval
This Thesis titled
Development of a Biosensor for Methyl Parathion
Detection and Genetic Risk Assessment of Exposed
Population
by
Muhammad Idrees
CIIT/FA07-PBS-004/ISB
Has been approved
For the COMSATS Institute of Information Technology, Islamabad
External Examiner 1: __________________________________________
External Examiner 2: __________________________________________
Supervisor: ______________________________________________
Dr. Syed Habib Ali Bokhari
Professor & Chairman, Dept of Biosciences, CIIT Islamabad
HoD: __________________________________________________
Dr. Raheel Qamar (T.I.)
Professor, Dept of Biosciences, CIIT Islamabad
Chairman:
Dr. Syed Habib Ali Bokhari
Professor, Dept of Biosciences, CIIT Islamabad
Dean, Faculty of Science: ___________________________________
Dr. Arshad Saleem Bhatti (T.I.)
Professor, Dept of Physics, CIIT Islamabad
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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
CIIT/FA07-PBS-004/ISB
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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:
________________________
Dr. Syed Habib Ali Bokhari
Professor& Chairman
Department of Biosciences
Head of Department:
________________________
Dr. Raheel Qamar
Professor
Head, Department of Biosciences, CIIT, Islamabad
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DEDICATION
To Almighty ALLAH and the Holy Prophet
Muhammad (P.B.U.H)
&
My Teachers, Parents & Family
ACKNOWLEDGMENTS
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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.
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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
CIIT/FA07-PBS-004/ISB
`
ABSTRACT
Methyl parathion is one of the highly toxic organophosphate pesticides which contaminates
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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
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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
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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
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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
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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
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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
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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
5.3.2. Serum arylesterase activities according to PON1 L55M and PON1 Q192R-
------------------------------------------------------------------------------------------------99
5.3.3. Serum arylesterase activities according to PON1 L55M and PON1 Q192R-
---------------------------------------------------------------------------------------------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
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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
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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
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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
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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
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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
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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
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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
Fig. 4.5: Comparison of arylesterase activity between cases (n = 10) and healthy controls
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
of family (n = 24) and healthy Punjabi population (n = 140) with respect of combined
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
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Fig. 5.2: Diagram showing normal structure of eye with normal fluid flow which leads to
normal IOP (Intra Ocular Pressure) for normal eye function --------------------------------87
Fig. 5.3: Diagram showing normal structure of eye with normal fluid flow which leads to
normal IOP (Intra Ocular Pressure) for normal eye function --------------------------------87
Fig. 5.4: Diagram showing normal structure of eye with normal fluid flow which leads to
normal IOP (Intra Ocular Pressure) for normal eye function --------------------------------88
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
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. ---------------------------------------------------------------------------101
Fig. 5.12: Comparison of serum paraoxonase activities (U/L) of Glaucoma patients (PCAG
and POAG) with normal controls and healthy male Punjabi population according to
combined genotypes -------------------------------------------------------------------------102
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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
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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
Table 3.2: Allele and genotype frequencies of coding region polymorphisms of PON1
gene in healthy Pakistani Punjabi pesticide sprayers (n=100). ---------------------------50
Table 3.3: Allele and genotype frequencies of coding region polymorphisms of PON1
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
Table 4.2: Allele and genotype frequencies of coding region polymorphisms of PON1
gene in healthy controls of Punjabi hypercholesterolaemia family (n=24)--------------72
Table 4.3: Allele and genotype frequencies of coding region polymorphisms of PON1
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
Table 5.3: Allele and genotype frequencies of coding region polymorphisms of PON1
gene in age and gender matched healthy controls (non-glaucomic) (n=70).----------- 94
Table 5.4: Allele and genotype frequencies of coding region polymorphisms of PON1
gene in POAG patients (n=90). ---------------------------------------------------------------95
Table 5.5: Allele and genotype frequencies of coding region polymorphisms of PON1
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
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
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
2.3.6. Serum arylesterase activities according to PON1 L55M and PON1 Q192R
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).
Table 3.2: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
Table 3.3: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
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
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).
3.3.10. Serum arylesterase activities according to PON1 L55M and PON1 Q192R
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
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
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
3.3.11. Serum paraoxonase activities according to PON1 L55M and PON1 Q192R
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
/LM
/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
/LM
/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
Table 4.2: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
Table 4.3: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
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
Punjabi healthy
population (n = 140)
cxvi
Fig. 4.2: Comparison of combined genotype frequencies between healthy Punjabi
population, normal healthy controls and hypercholesterolaemia patients
4.3.4. Serum arylesterase activities according to PON1 L55M and PON1 Q192R
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
LL LM MM RR Q
R QQ
0
10
20
30
40
50
60
70
80
Prevalence (%)
PON1 L55M
PON1
Q192R
RR
QR
Healthy members
of studied Punjabi
family (n = 24)
Hypercholesterolaemia
patients of studied
Punjabi family (n = 10)
cxvii
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. a: statistically significant differences between patients and
normal controls of the family. b: statistically significant differences between patients and
healthy Punjabi population.
4.3.5. Serum paraoxonase activities according to PON1 L55M and PON1 Q192R
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
4.3.6. Serum arylesterase activities according to combined genotypes
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.
Fig. 4.5: Comparison of arylesterase activity between cases (n = 10) and healthy controls
of family (n = 24) and healthy Punjabi population (n = 140) with respect of combined
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
/LL
/LM
/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
Control Patients Healthy Punjabis
a, b
a
b
cxx
4.3.7. Serum paraoxonase activities according to combined genotypes
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
of family (n = 24) and healthy Punjabi population (n = 140) with respect of combined
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
/LL
/LM
/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
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
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.
(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).
Fig. 5.2: Diagram showing normal structure of eye with normal fluid flow which leads to
normal IOP (Intra Ocular Pressure) for normal eye function
(http://www.webeca.com/content/vision%20problems/Glaucoma.htm)
cxxx
Fig. 5.3: Diagram showing normal structure of eye with normal fluid flow which leads to
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).
cxxxi
Fig. 5.4: Diagram showing normal structure of eye with normal fluid flow which leads to
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).
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
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
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%).
Table 5.3: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
Table 5.4: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
Table 5.5: Allele and genotype frequencies of coding region polymorphisms of PON1 gene
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
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
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
population (n = 140)
cxlii
Fig. 5.8: Comparison of genotypes between Glaucoma patients (PCAG and POAG) with
normal controls and healthy male Punjabi population
5.3.2. Serum arylesterase activities according to PON1 L55M and PON1 Q192R
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
LL LM MM RR Q
R QQ
0
5
10
15
20
25
Prevalence (%)
PON1 L55M
PON1
Q192R
RR
QR
LL LM MM RR Q
R QQ
0
5
10
15
20
25
30
Prevalence (%)
PON1 L55M
PON1
Q192R
RR
QR
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.
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. 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
5.3.3. Serum paraoxonase activities according to PON1 L55M and PON1 Q192R
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).
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. 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
5.3.4. Serum arylesterase activities according to combined genotypes
-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
/LL
/LM
/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
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
5.3.5. Serum paraoxonase activities according to combined genotypes
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).
Fig. 5.12: Comparison of serum paraoxonase activities (U/L) 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
-50
50
150
250
350
450
550
650
/LL
/LM
/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
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. Materials and Methods
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
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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
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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).
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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).
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OP: Organophosphate compound,
FP-biotin: 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide,
DFP: Didisopropylfluorophosphate,
CPO: Chlorpyrifos oxon,
OP-Tyr: Tyrosine residue at which OP binds
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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
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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.
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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.
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7.2. Materials and Methods
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)
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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
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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.
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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
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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:
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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
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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)
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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
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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.
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Fig. 7.5: Neighbor joining tree of Anthozoan fluorescent proteins using nucleotide
sequences of genes.
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Fig. 7.6: Neighbor joining tree of Anthozoan fluorescent proteins using amino acid
sequences.
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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.
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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)
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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.
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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
Primer sequence (5’-3’) TA Product
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
Primer sequence (5’-3’) TA Product
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)
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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.
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