Lead levels in teeth as a measure of life-time lead exposure in children Charuwan Manmee Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Medical Sciences Institute of Health and Society 2012 ABSTRACT Background and aims Lead exposure has irreversible health effects in children who are susceptible even at very low levels of exposure. The usual test for lead exposure is blood lead level (BLL), but this indicates only recent exposure. This study aimed to ascertain the suitability of milk teeth as biomarker of the history of lead exposure and to develop a methodology for this novel biomarker. Methods My study comprised three stages: Firstly, I explored potential determinants of dentine lead levels (DLLs) in children living in Newcastle upon Tyne (the Tooth Fairy Study). Secondly, I developed a methodology for assessing the history of early life lead exposure using dentine, and thirdly I applied my methodology to newly extracted teeth from children in Teesside. The Tooth Fairy study cohort consisted of 69 children aged 5-8 years. DLLs were measured in primary dentine using Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To identify determinants of early life exposure, a questionnaire was used. I assessed associations between lifestyle characteristics and DLLs. As teeth develop chronologically they offer the opportunity to study histories of exposure in detail. I collected two deciduous molars each from 15 children aged 6-8 years living in Northeast England. By combining high spatial resolution LA-ICP-MS with dental histology, I acquired information on the age specific concentrations of lead in dentine from in utero to several years after birth. Results Dentine lead levels in the Tooth Fairy cohort ranged from 0.06 to 0.77µg/g, median 0.21µg/g. Unlike other studies, I did not find significant associations between socio- economic status or other possible determinants and lead exposure. ii In developing the biomarker I found that the 100 micron ablation pit represented 42 days of dentine growth, enabling me to assign an age interval to each ablation pit. I found lead ratios in primary dentine to be consistent between teeth from the same child, and at the same age within each tooth. This indicated that the history of exposure can be determined using a single, multi-point ablation transect on high quality longitudinal sections of individual teeth. Conclusions I found no association between socio-economic status and dentine lead levels in a cohort of children from Newcastle upon Tyne. I developed a novel technique to date ablation points in dentine in milk teeth, and, using this technique have demonstrated that primary dentine is a potential biomarker for characterising the early life history of lead exposure in children. iii ACKNOWLEDGEMENTS It has always been my dream to study for a PhD degree. My dream came true on the day I started as a PhD student at Newcastle University. It is difficult to express my appreciation for the people who have me supported throughout my time here. The foremost persons that I would like to thank are my supervisors; Dr Susan Hodgson, Professor Tanja Pless- Mulloli, Dr Wendy Dirks and Dr Thomas Shepherd. My sincere appreciation gives to all of them for their enthusiasm, support, advice and encouragement throughout my study. In particular, Dr Susan Hodgson advised me on every aspect of the study, whose conversation both academic and non-academic, has helped me in many ways. She never showed any signs of being tired of my questions, requests and problems. She has been like my older sister in guiding me through my study. I also thank Professor Tanja Pless- Mulloli for giving me direction and support, and for her insights and invaluable advice which have helped me to reach this milestone. I am grateful for the opportunity to have been a student in her team. Dr Wendy Dirks deserves heartfelt thanks for encouraging me to explore new histological techniques, provide me the opportunity to learn so much in a way that few people have done so far. Her extensive knowledge on the histology of teeth enabled me to carry out the analysis of the samples and interpret the findings. My sincerest thanks go to Dr Thomas Shepherd for sharing his knowledge of analytical techniques, and for the time he spent to help in analysing the data. My experience at Leeds has also been in my thoughts. I believe that without all of my supervisors I would have had no clear direction to my research, and would not have been able to complete this study. As such, I am most grateful for their encouragement and persistence. I would like to convey thanks to Professor Jimmy Steele and Dr Caroline Relton for their valuable advice during my annual assessments. I am grateful to my colleagues at the Institute of Health and Society (IHS) for their support and encouragement over the years. I am particularly grateful to my friends, especially Stephanie O’Neil, Jenan Shakoor and Nor Farahidah Za'bah, for reading my drafts and offering invaluable advice as well as sharing knowledge and experience with me. I wish to thank Chon Poosuwan for the time he spent to help in the Geographic Information System (GIS), and the staff of the IHS for their kind assistance and support during these years. Many other people are not mentioned here by the name but they too are always in my thoughts. iv Special thanks to Dr Paul Averley and his colleagues from the Queensway Dental Practice Clinic in Billingham who supported the study and facilitated both recruitment and sample collection. I wish to acknowledge and thank the expert laboratory assistance of Pam Walton for her help in preparing samples. She never showed any sign of being tired of my needs and always helped as much as she could. I would like to thank the Department of Medical Services, Ministry of Public Health, Thailand, for the grant which paid for my studies. I acknowledge the partial funding support from the IHS and the centre for Oral Health Research, School of Dental Sciences, Newcastle University, for laser ablation analyses. I would like to express my gratitude to my whole family, for their belief in me. To my parents, thank you for encouraging me to always do my best. You have always been supportive of my endeavours and proud to celebrate my accomplishments. Without you, I would not be the person I am today. To my older sisters, thank you for your confidence and friendship. You have provided me with much unconditional love, support and care. I extend my total love to my beloved relatives and colleagues in Thailand, who have always helped, supported and believed in me. v STATEMENT OF CONTRIBUTION This is to certify that the work contained in this thesis comprises original work conducted by the student under supervision of Professor Tanja Pless-Mulloli, Dr Susan Hodgson, Dr Wendy Dirks and Dr Tom Shepherd. My own contributions and those of others were as follows: For the epidemiological study I used survey data collected prior to my arrival. I carried out all statistical analysis of these data. Dr Paul Averley and his team at Queensway Dental Clinic assisted in the recruitment process and with sample collection of teeth and blood samples in Teesside. Blood samples were analysed at the Health and Safety Laboratory, Buxton. I used the blood lead data for my analysis For the histological study I carried out all measurements. For the measurement of lead I prepared the teeth with the assistance of Pam Walton in the School of Dental Sciences, Newcastle University. The lead analysis was carried out by Dr Tom Shepherd and his team at the University of Leeds. I used all data to interpret with the histological dating. The thesis has not been submitted for the award of any other degree at any other institution. vi JOURNAL PUBLICATIONS AND CONFERENCE PAPERS 1. Shepherd TJ, Dirks W, Manmee C, Hodgson S, Banks DA, Averley P, Pless-Mulloli T. Reconstructing the life-time lead exposure in children using dentine in deciduous teeth. Science of the Total Environment. 2012;425:214-22. 2. Manmee C, Hodgson S, Dirks W, Shepherd T, Pless-Mulloli T. (2011) Lead levels in teeth as a measure of life-time lead exposure in children. American Journal of Physical Anthropology, 2011: 144, Supplement 52:205. Presented at The 2011 AAPA Symposium in Honor of Don Reid. Evolutionary Anthropology. 2011; 20:161-163. (Poster presentation, 80th Annual meeting of the American Association of Physical Anthropologists (AAPA), Minneapolis, Minnesota, USA, April 2011) 3. Manmee C, Hodgson S, Dirks W, Shepherd TJ, Pless-Mulloli T. Lead levels in teeth as a measure of life-time lead exposure in children. Abstracts of the 23rd Annual Conference of the International Society of Environmental Epidemiology (ISEE). September 13 - 16, 2011, Barcelona, Spain. Environ Health Perspect :- http://dx.doi.org/10.1289/ehp.isee2011. (Poster presentation, 23rd Annual Conference of the International Society for Environmental Epidemiology (ISEE), Barcelona, Spain, September 2011) 4. Manmee C, Hodgson S, Dirks W, Shepherd TJ, Pless-Mulloli T. Dentine lead levels in children in Newcastle upon Tyne. Abstracts of the 23rd Annual Conference of the International Society of Environmental Epidemiology (ISEE). September 13 - 16, 2011, Barcelona, Spain. Environ Health Perspect :-. http://dx.doi.org/10.1289/ehp.isee2011. (Poster presentation, 23rd Annual Conference of the International Society for Environmental Epidemiology (ISEE), Barcelona, Spain, September 2011) 5. Manmee C, Hodgson S, Dirks W, Shepherd TJ, Pless-Mulloli T. Lead levels in teeth as a measure of life-time lead exposure in children (Oral presentation, Annual conference of the North East Postgraduate Conference (NEPG), Newcastle upon Tyne, UK, November 2010) 6.