19/03/2012 Toxicologicalcharacterizationof PM10: case study Genk-Zuid. R. Van Den Heuvel
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1 19/03/2012 Toxicologicalcharacterizationof PM10: case study Genk-Zuid. R. Van Den Heuvel
2 Introduction» Assessment of potential risk to human health associated with environmental air» Fysico-chemical characterization» Limitations» Known chemicals» Number of measured chemicals» Mixture toxicity» Evaluation of air quality using effect-oriented measurements» Biological tests with specific mechanism of action» Genotoxicity, immunotoxicity, endocrine disruption,» Hazard identification» Relation to human health not clear 19/03/2012 2
3 Goal In order to better assess the potential risk to human health from environmental air quality, effect-oriented measurements will be performed simultaneously with human biomonitoring of the local population in the selected hotspot Genk-Zuid from the Flemish Centre of expertise on Environment and Health(Steunpunt Milieu en Gezondheid). Toxicological characterization of air in Genk-Zuid using effect-oriented measurements Estimate possible health risk Health effects in HBM 19/03/2012 3
4 Hot spot Genk-Zuid Flemish Center for Expertise on Environment and Health HBM : reference values for large number of pollutants Status of the general population Control group for selected hotspots : hot spots in Flanders Biomonitoring in a selected area January2010 -November 2010 : Genk-Zuid 19/03/2012 4
5 Hot spot Genk-Zuid Industrial zone» Industries: stainless steel, car production, chipboard, power station (coal, biomass)» High traffic density Environmental monitoring» heavy metals, PCB126, dioxin (dust, soil, vegetables) Population Health survey (questionnaires) (2007): Complaints: traffic, noise, air pollution, odour Health problems: airway problems, antidepressant medication 19/03/2012 5
6 Hot spot Genk-Zuid: HBM» Recruitment 200 adolescents» Selectionof biomarkers:» pollutants: basedin inventoryof industryandenvironmentalmonitoring (heavy metals, POPs, PAH, benzene, toluene) associated health effects» Genk-Zuid vs. Flemish reference group 19/03/2012 6
7 Strategy measuring campaign» The measuring strategy was tuned with the human biomonitoring strategy results of biomarkers will be compared with referencevaluesfrom Flemish reference biomonitoringcampaign» Choice of the measuring locations» Genk-Zuid» Flemish reference area: Koksijde 19/03/2012 7
8 Strategy measuring campaign» Sampling» Measuring campaign 6 months: January 2010 July 2010» Frequency: tuned HBM» Biomarkers of effect medium/long term exposure» Air sampling in Genk and Koksijde» spread, ±every 3 day, 24h sample» One pooled sample per month Maand Koksijde Genk Februari Maart April 9 13 Mei 6 11 Juni 8 10 Juli - 10» Biomarkers of effect recent exposure» Air sampling in Genk (24h): day preceding medical examination day» 10 one-day samples (January till June 2010) 19/03/2012 8
9 Strategy measuring campaign» Sampling of PM10» Sampling device» Genk» 3x Low volume sampler PartisolPlus type 2025» Teflon filter (PTFE, Teflo, PALL Life Sciences,Ø=47mm)» Koksijde» High volume sampler DigitelDHA-80 sampler» Teflon filter (PTFE, T38,Schleicher-Schull, Ø=150mm)» Filter treatment» Pooled samples per month» Organic extraction (ASE)» Particles» Day samples» Particles 19/03/2012 9
10 Selection of tests» HBM Health effect HBM Biomarker Carcinogenic effects Immune effects Endocrine disruption Neural effects Renal effects Comet assay Questionnaire asthma and allergy Thyroid, puberty, sex hormones NES test, ADHD Beta2-microglobuline, cystatine-c» Genotoxicity: Ames test» Bacterial test (Salmonella typhimuriumhis - reverse mutation test ): strain TA98» Most used and validated test in genotoxicity» Genetically modified strain: His -» Mutagenicity: backmutationsin histidine-operon(his - His + )» + and S9 (metabolic rat lever fraction)» Reference compound: B(a)P 19/03/
11 Selection of tests» Immune toxicity: cytokine (il-6 and il-8) induction in Beas-2B cells» Beas-2B human bronchial epithelial cell line» Pro-inflammatory cytokines» Il-6: activation immune response by stimulation of lymphocytes» Il-8: chemokine, attracks target cells (e.g., neutrophil granulocytes) to inflammation site» Reference compound: LPS (endotoxin)» Endotoxin test» component of the cell wall of gram negative bacteria» illicit a strong response in the immune system e.g. cytokine production» Present on particles» LAL-testkit 19/03/
12 Selection of tests» Endocrine disruption: MELN assay» Humane breast cancer MCF-7 cell line,» stable transfection with estrogen receptor (ERE-βGlob-Luc-SVNeo)» estrogen activities: luciferasesignal following receptor binding» Detection of agonist en antagonist activity» Reference compound: estrogen ER antagonists Xeno-estrogens agonists ERE Luciferasegene RNA Luciferase Light» Systemic toxicty: radical generating capacity» Biological damage 19/03/
13 Selection of tests Health effect In vitro test Particles/ Air sample extract Genotoxicity Ames ASE extract Month samples Immune toxicity Beas-2B bronchial cell line: il-6/il-8 particles Month samples Endotoxine test particles Month samples Endocrine disruption MELN test ASE extract Month samples Systemic toxicity Cytotoxicity inbeas-2b particles Month samples Radical generating potency particles Day samples» ASE extract: exposure to day-equivalent(amount inhaled air /24 hours)» normal breathing during 24 h gives an equivalent 20 m 3 /day» Particles: exposure on basis of mass (100, 50, µg PM10/ml medium)» Concentration range, positive/negative controls, reference compound 19/03/
14 Results» Trend in function of time» Trend in function of location» Relative toxicological potency» Toxicological profile of Genk-Zuid and Koksijde 19/03/
15 Ames revertanten Koksijde Ames -S m 3 luchtequivalent Februari Maart April Mei Juni revertanten Koksijde Ames +S m 3 luchtequivalent Februari Maart April Mei Juni 200 Genk Ames -S9 200 Genk Ames +S revertanten revertanten m 3 luchtequivalent Februari Maart April Mei Juni Juli m 3 luchtequivalent Februari Maart April Mei Juni Juli 19/03/
16 il-6 en il-8 50 Koksijde il-6 90 Genk il-6 (pg/ml) il-6 (pg/ml) PM10 (µg/ml) Blanco Februari Maart April Mei Juni PM10 (µg/ml) Blanco Februari Maart April Mei Juni Juli 800 Koksijde il Genk il-8 pg/ml il-8 pg/ml PM10 (µg/ml) Blanco Februari Maart April Mei Juni PM10 (µg/ml) Blanco Februari Maart April Mei Juni Juli 19/03/
17 MELN-test 800 Koksijde (1) 800 Genk (1) Luciferase activiteit (% inductie) Luciferase activiteit (% inductie) concentratie (m 3 luchtequivalent) Februari Maart April Mei Juni concentratie ( m 3 luchtequivalent) Februari Maart April Mei Juni Juli 19/03/
18 Radical generating capacity» No trend in time» Trend in function of location * 10 day samples Genk 53 day samples Koksijde» Strong positive association with cytokine induction» il-6: r= 0.88, p=0.002 (n=9)» Il-8: r=0.8, p=0.01 (n=9) * t-test, p<0.01) 19/03/
19 Results» Toxicological profile of Genk-Zuid and Koksijde» Toxic response expressed as equivalent of reference compound RTE (Relative toxicological equivalent) Cytotoxiciteit MELN 1 0,8 0,6 0,4 0,2 0 * il-6 * Interne controle Genk * Ames +S9 il-8 * Koksijde Ames -S9 * 5 month samples per location (Febr till July 2010) * Significant difference between Genk and Koksijde (Repeated measures ANOVA, p<0.05) 19/03/
20 Relation effect oriented measurements and human health in Genk-Zuid» Relation between in vitro results and biomarkers medium/long-term exposure exposure In vitro results Genotoxicity Ames test Mutagenic effect Health effect in HBM comet assay: significantly higher (+26%) 19/03/
21 Relation effect oriented measurements and human health in Genk-Zuid» Relation between in vitro results and biomarkers medium/long-term exposure exposure In vitro results Genotoxicity Ames test Mutagenic effect Health effect in HBM comet assay: significantly higher (+26%) Immune toxicity Il-6/il-8 Respiratory inflammation Asthma and allergy less allergy for animals more asthma in some districts Health survey Airway problems 19/03/
22 Relation effect oriented measurements and human health in Genk-Zuid» Relation between in vitro results and biomarkers medium/long-term exposure exposure In vitro results Genotoxicity Ames test Mutagenic effect Health effect in HBM comet assay: significantly higher (+26%) Immune toxicity Il-6/il-8 Respiratory inflammation Endocrine disruption MELN test estrogenic activity Asthma and allergy less allergy for animals more asthma in some districts Health survey Airway problems Endocrinology: thyroid, puberty differences, but normal ranges 19/03/
23 Relation effect oriented measurements and human health in Genk-Zuid» Relation between radical generating capacity and biomarkers recent exposure» Association with biomarker of oxidative DNA damage in comet assay» r=0.2, p=0.02» Correction gender, age, BMI, smoking, PM10 mass n=126 adolescents Genk 19/03/
24 Conclusion» The study showed that a toxicological profile of the collected PM10 can be determined by the used biological tests.» The mutagenic, inflammatory, endocrine disrupting, and radical generating capacity of the air samples varied in function of time, location.» The measured endpoints were significantly increased in Genk compared to Koksijde.» The simultaneous performance of effect-oriented measuring and human biomonitoring of the local population in the selected hot spot Genk-Zuid allowed for a better relationshipbetween and estimationof air quality and possible related health risks.» The health risk was well estimated using effect-oriented measurements. 19/03/
25 Acknowledgement» FinanciallysupportedbyFlemish Government, Department of Environment, Nature and Energy» VITO» H. Witters» N. Bleux» D. Ooms» H. Leppens» P. Berckmans» J. Maes» University Hasselt» T. Nawrot» B. Cox» Flemish Government» M. Mampaey» K. Van Campenhout 19/03/
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