Paraoxonase (PON1) polymorphisms as a biomarker of susceptibility to organophosphate toxicity among a cohort of singaporean workers

PARAOXONASE (PON1) POLYMORPHISMS AS A BIOMARKER OF SUSCEPTIBILITY TO ORGANOPHOSPHATE TOXICITY AMONG A COHORT OF SINGAPOREAN WORKERS SAFIYYA MOHAMED ALI B.Sc Life Sciences (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF COMMUNITY, OCCUPATIONAL AND FAMILY MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my utmost gratitude to my supervisor Professor Chia Sin Eng who proposed PON1 for me as a subject of this Masters thesis During my experience as a Research Assistant and a Masters student in the Department of Community, Occupational and Family Medicine (COFM), Yong Loo Lin School of Medicine, National University of Singapore, Prof Chia has introduced me to the world of occupational health, neurotoxicity, statistics and epidemiology Prof Chia has not only been an invaluable guiding and motivational source in my academic pursuit, but also an important source of support for my personal development His kindness and dedication to his career as an occupational health physician and a researcher have inspired me greatly in the field of public health I express my thanks to Professor Chia Kee Seng from the Centre of Molecular Epidemiology, NUS, for organising the courses under the NUS-KI Joint PhD programme in Genetic and Molecular Epidemiology (GAME) and giving me the opportunity to attend the modules that were conducted in Sweden He has also given constructive criticism of the project which helped me to look into various angles of the analysis Special acknowledgements to him and Dr Teo Yik Ying of the Singapore Genome Variation Project for early access to unpublished data on the allele frequency of rs662 (PON1Q192R) across the three ethnic groups in Singapore, and more specifically to Miss Sim Xueling for her help in extracting this data I am grateful to our collaborator Professor Eric Yap Peng Huat for giving me access to carry out genotyping work at the Defence Medical and Environmental Research Institute laboratory, Defence Science Organisation, Singapore This portion of the project would not have been possible without the help of Ms Rachel Tham, Ms Linda Gan and Mr Yim Onn Siong who lent me their expert knowledge in the area i I am thankful to my co-workers namely Mr Ong Her Yam, Mr Ong Yeong Bing, Mr Andrew Wee, Ms Vivian Ng, Ms Chua Lay Ha, Ms Amy Chan and Ms Julie Chew They were involved in the field trips for the medical surveillance of all the workers and were responsible for the collection of the study materials I also deeply appreciate the technical help from Mr Ong Yeong Bing for brainstorming and optimising enzymatic assays with me He has always been ready to lend a hand whenever I needed help in the laboratory My sincere thanks are due to Ms Lim Gek Hsiang for her assistance and advice in the statistical analysis of this thesis and of our published original article The numerous scientific and less scientific discussions with her have created a pleasant working atmosphere and a wonderful friendship Warm thanks go to my parents, sister, and the rest of my family for their understanding, support and comfort not only during this project, but throughout my previous studies I would like to dedicate this thesis to my dear fiancé Farooq; his love and support has kept me going especially on days where everything seemed to be going wrong Because of him, I had the strength and motivation to complete this thesis Finally and most importantly, all praises be to God who granted me the opportunity and strength to undertake my Masters studies We would like to thank the men who have participated in the study This study was supported in part by a grant from the National Medical Research Council, NMRC/EDG/0008/2007 and the Life Sciences Institute, National University of Singapore, R-329-000-008-712 ii PUBLICATIONS Papers published and accepted for publication in international journals Chia Sin Eng, Safiyya Mohamed Ali, Yap Peng Huat Eric, Linda Gan, Ong Yeong Bing, Chia Kee Seng Distribution of PON1 polymorphisms – PON1Q192R and PON1L55M among Chinese, Malay and Indian males in Singapore and possible susceptibility to organophosphate exposure Neurotoxicology 2009 (Accepted for publication) Safiyya Mohamed Ali and Sin Eng Chia Interethnic variability of plasma paraoxonase (PON1) activity towards organophosphates and PON1 polymorphisms among Asian populations – A Short Review Industrial Health 2008; 46(4):309 Papers presented at international meetings Safiyya Mohamed Ali, Sin Eng Chia Association between cholinesterase Activity and human paraoxonase (PON1) polymorphisms in workers exposed to organophosphates HUGO 13th Human Genome Meeting (HGM2008), 27-30 September 2008, Hyderabad, India Sin Eng Chia, Safiyya Mohamed Ali, Yeong Bing Ong Paraoxonase (PON1) activities and genotype distribution among Chinese, Malay and Indian workers in Singapore 20th International Conference on Epidemiology in Occupational Health (EPICOH-NEUREOH 2008), 9-11 June 2008, Heredia, Costa Rica iii Safiyya Mohamed Ali, Sin Eng Chia Interethnic variability of plasma paraoxonase (PON1) activity and PON1 polymorphisms among Asian populations The 5th Princess Chulabhorn International Science Congress (PCVI), Chulabhorn Research Institute, 25-29 November 2007, Bangkok, Thailand Sin Eng Chia, Safiyya Mohamed Ali Association of RBC and serum cholinesterase levels among organophosphate exposed pesticide workers and noexposed workers in Singapore The 5th Princess Chulabhorn International Science Congress (PC-VI), Chulabhorn Research Institute, 25-29 November 2007, Bangkok, Thailand iv TABLE OF CONTENTS SUMMARY viii LIST OF TABLES .x LIST OF FIGURES .x INTRODUCTION LITERATURE REVIEW Organophosphates .4 Structure and properties of organophosphates Applications of organophosphates Organophosphate toxicity Cholinesterase and organophosphate toxicity Paraoxonase-1 (PON1) Protein .11 Introduction to the PON1 protein 11 Synthesis and structure 13 Measurement of activity 14 Measurement of concentration 17 Role of PON1 in detoxifying organophosphates 17 PON1 Gene 20 PON gene family and structure of PON1 20 PON1 polymorphisms 21 Coding region polymorphisms: Q192R and L55M 22 Promoter region polymorphisms .24 PON1 haplotypes .24 PON1 polymorphisms and ethnic distribution .25 PON1 and Organophosphate Toxicity 28 PON1 levels and polymorphisms .28 Environmental factors affecting PON1 concentration and activity .30 v AIMS OF THE STUDY 33 PARTICIPANTS AND METHODS 35 Study Population 35 Laboratory Methods 36 Blood sampling 36 Cholinesterase measurements 37 PON1 activity measurements 37 PON1 polymorphism screening .38 Statistical methods 40 RESULTS 42 Descriptive Characteristics of the Study Population 42 Cholinesterase Measurements 46 Cholinesterase levels between exposure groups 46 Cholinesterase levels between ethnicities 46 Cholinesterase levels between groups, stratified by ethnicity 47 Predictors of cholinesterase levels 47 PON1 (Paraoxonase/Diazoxonase) Activity 48 PON1 activity between exposure groups 48 PON1 activity between ethnicities 48 PON1 activity between exposure groups, stratified by ethnicity .49 PON1 Genotypic Distribution 50 PON1 genotypic distribution by exposure groups 50 PON1 genotypic distribution by ethnicities .51 PON1 alleles 52 PON1 genotypic distribution by exposure groups, stratified by ethnicity .53 PON1 genotype and PON1 activity 54 PON1 Status .55 vi PON1 Genotypes and Cholinesterase Levels 56 Factors affecting PON1 activity 57 DISCUSSION .58 Study Participants 58 Methodological Considerations 60 Cross-sectional study .60 Questionnaire 60 Laboratory Methods .62 Cholinesterase Measurements 64 PON1 Activity, Status and Genotype .67 Recommendations Arising from the Study 71 Future Prospects 72 SUMMARY AND CONCLUSIONS 75 BIBLIOGRAPHY 78 APPENDICES 94 Appendix I 95 Appendix II .103 Appendix III .113 vii SUMMARY Organophosphate (OP)-containing pesticides are extensively used worldwide for domestic and industrial purposes Studies on acute and chronic exposure to OPs have revealed numerous health effects attributed mainly to acetylcholinesterase inhibition The enzyme human serum paraoxonase (PON1) is involved in the detoxification of OP compounds PON1 polymorphisms have been shown to affect susceptibility to OP exposure In this study, we investigated the effect of OP exposure on pest control workers using cholinesterase levels as biomarkers of toxicity Next we determined PON1 activity towards two OP substrates (paraoxon and diazoxon) of our sample as well as the distribution of two common PON1 polymorphisms in our local population (PON1Q192R and PON1L55M) and assessed whether these factors were involved in OP toxicity The exposed group consisted of 103 workers from various pest control companies under the Singapore Pest Management Association while the 91 unexposed workers were from a lead stabiliser factory For all workers, the mean age was 36.9 (20–70) years and the ethnic distribution was 38.1% Chinese, 44.3% Malay and 17.5% Indian The mean ± SD exposure duration among the pesticide workers was 10.4 ± 8.4 years The mean ± SD erythrocyte cholinesterase level was 18436.2 ± 2078 U/L and 18079.6 ± 1576 U/L for the exposed and unexposed groups respectively (p=0.216) The mean ± SD serum pseudocholinesterase level was 11028.4 ± 2867.4 U/L and 9433.6 ± 2022.6 U/L in the exposed and unexposed groups respectively (p[...]... between ageing and toxicity, with certain agents like soman rapidly increasing ageing (Karalliedde 1999) 10 Paraoxonase- 1 (PON1) Protein Introduction to the PON1 protein Human serum paraoxonase (PON1) is a member of the A- esterase family (EC 3.1.8.1) that includes PON2 and PON3 Human PON2 and PON3 are similar to PON1 in that they are able to hydrolyse aromatic and long-chain aliphatic lactones (Draganov... compounds, aromatic carboxylic acid esters and carbamates (La Du 1992) This includes phenylacetate which measures PON1’s 16 arylesterase capabilities, toxic oxon forms of chlorpyrifos and the nerve agents sarin and soman (Smolen et al 1991) Recently, it has been hypothesised that lactonase activity instead of arylesterase or organophosphate activity could be a common feature of PON1 proteins (Draganov et al... This came about after the characterisation of the hydrolytic activity of PON1 on 30 lactones, thiolactones and cyclic carbonate esters (Billecke et al 2000; Draganov et al 2000; Jakubowski 2000) It is common convention to use the substrate name to define PON1 activity e.g paraoxonase if the substrate was paraoxon, “diazoxonase” if the substrate was diazoxon and “arylesterase activity” if the substrate... HDL peroxidation (Aviram et al 1998) It may also have a role in protecting plasma membranes from free radical injury (Durrington et al 2001) and degrades bioactive phospholipids like platelet-activating factor (PAF) (Rodrigo et al 2001) More recently, it was discovered that PON1 has lactonase activity and is involved in metabolism of drugs such as statins, spironolactone and glucocorticoid lactones (Billecke... substrate was phenylacetate Measurement of concentration Serum PON1 concentration can be measured using a competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (Blatter Garin et al 1994) or by sandwich ELISA using two monoclonal antibodies PON1 concentration has been shown to be positively associated with both serum arylesterase activity and serum paraoxonase activity (Kujiraoka... albumin-associated esterase activity will interfere with PON1 activity (Erdos and Boggs 1961) The assay has to be carried out in the presence of calcium as calcium has been shown to be important in maintaining the structural organisation of the PON1 protein as well as in regulation of its catalytic function (Kuo and Du 1998) PON1 status The method described above separates individuals into the three phenotypes of. .. exposure in Singapore An investigation into the exposure levels, using cholinesterase levels as biomarkers of OP toxicity, among our pesticide workers would give an important insight onto whether these regulatory measures are effective As an extension to this, it would be interesting to find out the different factors that might affect susceptibility to OP toxicity namely paraoxonase- 1 (PON1) levels,... exposure and rate of metabolic degradation In mild cases, symptoms are usually treated with oximes (e.g pralidoxime chloride) which reactivate phosphorylated AChE Treatment may be augmented with atropine which is an acetylcholine 7 antagonist that prevents the action of acetylcholine on muscarinic receptors (Namba et al 1971) Supplementary pharmacological treatment with diazepam and supportive treatment... OXItek Arylesterase /Paraoxonase Assay Kit (ZeptoMetrix Corporation, USA) Figure 5 Determination of PON1 status Plotting diazoxonase vs paraoxonase activities breaks the population into three groups, individuals homozygous at amino acid position 192 for Q (open circles) or R (open triangles) and heterozygotes (closed squares) which have been confirmed by PCR In addition, this analysis of hydrolysis rates... reaction of alcohols and phosphoric acid OPs are widely used as insecticides worldwide and they are readily available commercially for domestic and industrial purposes In addition, OPs are also utilised as therapeutic agents in the treatment of ophthalmic conditions (e.g echothiophate, isoflurophate) (Lloyd 1963) and as antihelmintics (trichlorfon) (Harder 2002), as well as nerve agents in chemical ... microplates and the OXItek Arylesterase /Paraoxonase Assay Kit (ZeptoMetrix Corporation, USA) Figure Determination of PON1 status Plotting diazoxonase vs paraoxonase activities breaks the population... beginning the assay) was used as an internal control A blank determination of basal assay mixture without serum was made to account for any spontaneous hydrolysis of diluted paraoxon/diazoxon solutions... activity e.g paraoxonase if the substrate was paraoxon, “diazoxonase” if the substrate was diazoxon and “arylesterase activity” if the substrate was phenylacetate Measurement of concentration Serum