BIOMONITORING OF ENVIRONMENTAL CONTAMINATION RESULTING FROM MINING ACTIVITIES ON EXPOSED POPULATIONS Patrícia Clara dos Santos Coelho Tese de doutoramento em Ciências Biomédicas 2013 Patrícia Clara dos Santos Coelho BIOMONITORING OF ENVIRONMENTAL CONTAMINATION RESULTING FROM MINING ACTIVITIES ON EXPOSED POPULATIONS Tese de Candidatura ao grau de Doutor em Ciências Biomédicas, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutor João Paulo Teixeira Categoria – Investigador Auxiliar Afiliação – Instituto Nacional de Saúde Dr. Ricardo Jorge Co-orientadora – Doutora Denisa Mendonça Categoria – Professora Associada Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Co-orientadora – Doutora Beatriz Porto Categoria – Professora Auxiliar Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto The experimental work presented in this thesis was performed at the: . Environmental Health Department, Portuguese National Institute of Health – Porto, Portugal; . Toxicology Unit, Department of Psychobiology, University of A Coruña – A Coruña, Spain; . Trace Element Laboratory, Faculty of Health and Medical Sciences, University of Surrey – Guildford, UK; . Department of Chemistry and Geochemistry, Colorado School of Mines – Golden, USA; . Division of Biological Chemistry, Biocenter Innsbruck Medical University – Innsbruck, Austria; . Department of Genetics, Faculty of Medical Sciences, New University of Lisbon – Lisbon, Portugal. Statistical analysis was performed by Dr. Blanca Laffon, Dr. Valentina Daal’Armi and Dr. Roberto Zoffoli under the orientation of Professor Dr. Stefano Bonassi at at IRCCS San Raffaele Pisana, Rome. This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the grant SFRH/BD/47781/2008, under the QREN - POPH - Type 4.1 - Advanced Training, subsidized by the European Social Fund and national funds from the MCTES. CONTENTS ORIGINAL PUBLICATIONS xi LISTS OF FIGURES xiii LISTS OF TABLES xv LIST OF ABBREVIATIONS xvii ABSTRACT xix RESUMO xxi I. REVIEW OF THE LITERATURE 1 1. Introduction 3 2. Mining Activities: Health Impacts 3 3. Panasqueira Mine: Study Area 4 3.1 Site description 4 3.2 Geology and mineralization 6 3.3 Mining activities and potential environmental considerations 8 3.4 Environmental studies: main results 10 4. Metal(loid)s 11 4.1 Human exposure 11 4.2 Toxicokinetics and bioaccumulation 17 4.3 Health effects 18 4.3.1 Genotoxicity, immunotoxicity, and carcinogenicity 18 5. Biomarkers: Human Health 20 5.1 Biomarkers of exposure 22 5.1.1 Biomarkers of internal dose 22 5.1.2 Biomarkers of biologically effective dose 23 5.2 Biomarkers of effect 24 5.2.1 T-cell receptor mutation assay 24 5.2.2 Micronuclei assay 25 5.2.3 Chromosomal aberrations 27 5.2.4 Comet assay 28 5.2.5 Immune markers 30 5.3 Biomarkers of susceptibility 34 5.3.1 Polymorphisms in genes involved in the metabolism (phase I and phase II) 35 5.3.1.1 GSTA2 35 5.3.1.2 GSTM1 36 5.3.1.3 GSTP1 37 vii 5.3.1.4 GSTT1 37 5.3.2 Polymorphisms in genes involved in DNA repair 37 5.3.2.1 Polymorphisms in genes involved in BER pathway 39 5.3.2.1.1 XRCC1 39 5.3.2.1.2 APEX1 39 5.3.2.1.3 MPG 40 5.3.2.1.4 MUTYH 40 5.3.2.1.5 OGG1 40 5.3.2.1.6 PARP1 41 5.3.2.1.7 PARP4 41 5.3.2.2 Polymorphisms in genes involved in NER pathway 41 5.3.2.2.1 ERCC family 41 II. AIM OF THE STUDY 43 III. MATERIAL AND METHODS 47 1. Study Population 49 1.1 Population selection 49 1.2 Study group 50 1.3 Sample collection 53 2. Biomarkers of Exposure 54 2.1 Metal(loid)s in blood, urine, nail and hair samples 54 2.1.1 Instrumentation 54 2.1.2 Reagents and standards 54 2.1.3 Sample preparation 54 2.1.4 Quality control and Quality assurance 55 2.1.5 Determination of creatinine 56 3. Biomarkers of Effect 57 3.1 T-cell receptor mutation assay 57 3.2 Cytokinesis Block MN Assay 57 3.3 Chromosomal Aberrations, aneuploidies and gaps 58 3.4 Comet Assay 58 3.5 Analysis of lymphocytes subsets 59 3.6 Quantification of neopterin, tryptophan, kynurenine, and nitrite 60 4. Biomarkers of Susceptibility 61 4.1 DNA extraction 61 4.2 Genotyping of polymorphisms in genes involved in the metabolism 61 viii 4.2.1 GSTA2 61 4.2.2 GSTM1 and GSTT1 61 4.2.3 GSTP1 62 4.3 Genotyping of polymorphisms in genes involved in DNA repair 62 4.3.1 XRCC1, APEX1, MPG, MUTYH, OGG1, PARP1 and PARP4 62 4.3.2 ERCC1, ERCC4 and ERCC5 63 5. Statistical Analysis 64 IV. RESULTS 67 1. Biomarkers of Exposure 69 1.1 Comparison with reference levels 70 1.2 Effect of exposure after adjustment for confounders 70 1.3 Correlations between matrices 73 1.4 Effect of gender, age and smoking habits 74 2. Biomarkers of Effect 76 2.1 Biomarkers of genotoxicity 76 2.1.1 Effect of exposure, age and smoking habits 76 2.1.2 Effect of gender 78 2.1.3 Synergistic effect of environmental and occupational exposure 79 2.1.4 Effect of metal(loid) concentration 79 2.2 Immune markers 81 2.2.1 Effect of exposure, age and smoking habits 81 2.2.2 Effect of gender 85 2.2.3 Effect of other confounders 85 2.2.4 Effect of metal(loid) concentration 86 2.3 Symptomatology 87 3. Biomarkers of Susceptibility 88 V. DISCUSSION 95 1. Biomarkers of Exposure 96 1.1 Comparison with reference levels 97 1.2 Correlations between matrices 97 1.4 Effect of gender, age and smoking habits 97 2. Biomarkers of Effect 100 2.1 Biomarkers of genotoxicity 100 2.1.1 Effect of metal(loid) concentration 100 2.1.2 Effect age and gender 101 ix 2.2 Immune markers 101 2.2.1 Effect of metal(loid) concentration 102 2.2.2 Effect of age and gender 104 2.3 Symptomatology 104 3. Biomarkers of Susceptibility 106 3.1 Polymorphisms in genes involved in the metabolism 106 3.2 Polymorphisms in genes involved in DNA repair 106 VI. CONCLUSIONS 111 VII. REFERENCES 115 VIII. ANNEXES 133 ANNEXE I 135 ANNEXE II 141 ANNEXE III 145 ANNEXE IV 149 ANNEXE V 153 ANNEXE VI 161 PUBLICAÇÕES DE SUPORTE À TESE DE DOUTORAMENTO 165 ACKNOWLEDGEMENTS 221 x ORIGINAL PUBLICATIONS The present work contains techniques and/or data also presented in the following scientific papers: • Coelho, P., García-Lestón, J., Costa, S., Costa, C., Silva, S., Fuchs, D., Geisler, S., Dall’Armi, V., Zoffoli, R., Bonassi, S., Pásaro, E., Laffon, B., and Teixeira, J.P. 2013. Immunological alterations in individuals exposed to metal(loid)s in the Panasqueira mining area, Central Portugal. Arch. Toxicol. (submitted). • Coelho, P., García-Lestón, J., Costa, S., Costa, C., Silva, S., Dall’Armi, V., Zoffoli, R., Bonassi, S., Lima, J., Gaspar, J., Pásaro, E., Laffon, B., and Teixeira, J.P. 2013. Genotoxic effects of environmental and occupational exposure to metal(loid)s. A molecular epidemiology survey of populations living and working in the Panasqueira mine area, Portugal. Envir. Health Perspect. (submitted). • Coelho, P., Costa, S., Costa, C., Silva, S., Walter, A., Ranville, J., Pastorinho, R., Harrington, C., Taylor, A., Dall'Armi, V., Candeias, C., Ferreira da Silva, E., Laffon, B., Bonassi, S., and Teixeira, J.P. 2013. Biomonitoring of several toxic metal(loid)s in different biological matrices from environmentally and occupationally exposed populations from Panasqueira mine area – Portugal. Environ. Geochem. Health (accepted, in press) • Coelho, P., Costa, S., Costa, C., Silva, S., Walter, A., Ranville, J., Sousa, A., Coelho, M., Garcia-Lestón, J., Pastorinho, R., Laffon, B., Pásaro, E., Harrington, C., Taylor, A., and Teixeira, J. 2012. Metal(loid)s levels in biological matrices from human populations exposed to mining contamination – Panasqueira Mine (Portugal). J. Toxicol. Environ. Health A 75: 893–908. • Coelho, P., García-Lestón, J., Silva, S., Costa, C., Costa, S., Coelho, M., Laffon, B., Pasáro, E., and Teixeira, J.P. 2011. Geno- and Immunotoxic effects on populations living near a mine - Case study: Panasqueira mine. J. Toxicol. Environ. Health A 74: 1076-1086. During these six years other scientific work was been published under to the same subject: • Coelho, P., and Teixeira, J. P. 2012. Biomarkers, Human Health. In Encyclopedia of Toxicology, 3rd Edition, Elsevier (in press). xi • Coelho, P., Teixeira, J.P., and Gonçalves O. 2011. Mining activities: Health impacts. In Encyclopedia of Environmental Health edit by Nriagu J.O. Volume 3, pp. 788-802, Burlington: Elsevier. • Coelho, P., Silva, S., Roma-Torres, J., Costa, C., Henriques, A., Teixeira, J., Gomes, M., and Mayan O. 2007. Health impact of living near an abandon mine – Case study: Jales Mines. Int. J. Hyg. Environ. Health 210: 399-402. xii LIST OF FIGURES Figure 1. (a) Location of Panasqueira mine (red arrow on the left map), the Castelo do Bode dam, and the Zêzere river flow (blue line across the map on the right); (b) Map showing the location of Rio tailing (~1,200,000m3) and mud impoundment (~731,034m3) (orange circle), Barroca Grande tailing (~7,000,000m3) and two mud impoundments (~1,193,885m3) (yellow circle) and several small villages nearby these structures (red squares: the one in the upper left corner is S. Francisco de Assis; the other three on the bottom belong to Barroca do Zêzere and Pampilhosa da Serra parishes). One can also see Zêzere river flowing from the middle right side, close to Rio tailings where it merges with a small river (Casinhas creek) coming from upstream Barroca Grande and that passes through S.