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Dioxines, PCB, Furanes et DR-CALUX

Présentation sur PCB, dioxines, furanes et analyses par DR CALUX (reporter gene) lors du GRMHST du 02.10.2014

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Dioxines, PCB, Furanes et DR-CALUX

  1. 1. Groupement romand de médecine, d’hygiène et de sécurité au travail Journée de présentation de cas 02 octobre 2014 Exposition aux PCB et Dioxines, qu’en pense le Dr CALUX ? Vincent PERRET Hygiéniste du travail certifié SSHT Hygiène du travail Toxicologie industrielle
  2. 2. Les PCB quelques points PolyChloroBiphenyles 209 congénères 2 • Liquides visqueux • Stables à la chaleur, inertes chimiquement • Isolant électrique • Très liposoluble et s’accumulent dans le long de la chaîne alimentaire • Perturbateurs endoctriniens • Cancérogènes probables (IARC 2a)
  3. 3. Nomenclature des PCB Nomenclature de Ballschmiter & Zell 3
  4. 4. Utilisation des PCB Usage des PCB répartition Condensateurs 50.3% Transformateurs 26.7% Plastifiants (joints) 9.2% Huiles hydrauliques 6.4% Papier carbone 3.6% Fluides caloporteurs 1.6% Additifs pétrolier 0.1% Autres 2.2% Usage industriel des PCB (1929-1975) – EPA 97 Production mondiale : 1.5 x 106 tonnes (UNEP 98). 4
  5. 5. Exemples d’application des PCB 5
  6. 6. Exemples d’application des PCB Joints de séparation (coupure) entre bâtiments Joints de raccordement Joints entre éléments Joints de retrait Joints dans bâtiment (1955-1975) < 200’000 ppm (20%) 6
  7. 7. Exemples d’application des PCB 7 Peintures industrielles (1955-1975) < 20’000 mg/kg (ppm)
  8. 8. Exemples d’application des PCB Peintures ignifuge de faux-plafonds 8
  9. 9. Exemples d’application des PCB Eléments bitumes d’étanchéité de toiture Source EPA 9
  10. 10. Exemples d’application des PCB 10 Eléments en amiante ciment Nombreux cas de contamination d’oeufs aux PCBs dans des fermes du nord de la Hollande et d’Allemagne
  11. 11. Effets sur la santé PCB et composés dioxin-like 11
  12. 12. Polychlorodibenzodioxines (PCDD) 75 congénères Polychlorodibenzofuranes (PCDF) 135 congénères Les PCB baby dioxine ? Polychlorobiphényls (PCB) 209 congénères 12 congénères planaires 12
  13. 13. Toxicité relative des composés dioxines, furanes et PCB-dl Facteurs d’équivalence toxique relatif au 2,3,7,8- TCDD 13
  14. 14. Les grand évènements impliquant PCBs et dioxines ¡ 1953 Ludwigsfhaven BASF ¡ 1960’ Vietnam, Agent Orange ¡ 1968 Yusho, Japon ¡ 1976 Seveso ICMESA ¡ 2004 Ukraine, Viktor Iouchtchenko 14 Liste non exhaustive, ne manquez pas le prochain épisode
  15. 15. SEVESO 15 A : Teq TCDD 15.5 – 580 μg/m2 B : Teq TCDD < 5 μg/m2 C : Teq TCDD < 1.5 μg/m2 Dégâts : - Fort impact sur végétaux et animaux (oiseaux) - Environ 200 cas de chloracné (88% enfants) - Modification du sex ratio dans la région (filles +) - Augmentation de cancer sujette à débat
  16. 16. Hamster Golden Syrian Face à la dioxine, qui est le plus fort ? Cochon d’inde Hartley 16
  17. 17. Toxicology Letters 230 (2014) 225–233 4.3. Controversy Although TCDD and the US National of debate regarding A completed report of PCDD TCDD was considered was not conclusive from an application clinical trials in from (1) some et al., 1982; Simon of other 2010; Brookes, between oncogenesis (Seifert Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Mini review AhR signalling and dioxin toxicity Olivier Sorg∗ University of Geneva Swiss Centre for Applied Human Toxicology (SCAHT), 1 rue Michel-Servet, 1211 Geneva 4, Switzerland O. Sorg / Toxicology Letters 230 (2014) 225–233 Table 2 Relation between TCDD LD50 and body fat precentage. Species (strain) Body fat [%] LD50 [!g/kg] Guinea pig (Hartley) 4.5 1 American dark mink 4 Hare 7.5 10 Chicken 35 Macaque 10 50 Rat (Sprague-Dawley) 10 50 Dog 100 Rabbit 10 120 Moiuse (C57BL) 8 150 Rat (Fischer) 10 300 Mouse (BALB/c) 400 Dog (Beagle) 14 1000 Frog 1000 Mouse (DBA) 20 2500 Hamster (golden Syrian) 15 10,000 h i g h l i g h t s • Besides its canonical pathway, AhR may activate other receptor-mediated one of the most resistant species with a LD50 of 12.5 mg/kg! How-ever, pathways. • AhR activity may be assessed by chemical or biological assays. • Dioxin toxicity cannot be explained only by AhR activation. • AhR activation leads to either upre-gulation or downregulation of genes. • TCDD as a human carcinogen is still a matter of debate. • NaturalAhRagonists found in vegeta-bles might have a beneficial effect. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 30 August 2013 Received in revised form 14 October 2013 Accepted 18 October 2013 Available online 12 November 2013 Keywords: Dioxin TCDD AhR Cell signalling Skin Toxicity a b s t r a c t Dioxins are a family of molecules associated to several industrial accidents such as Ludwigshafen in 1953 or Seveso in 1976, to the Agent Orange used during the war of Vietnam, and more recently to the poisoning of the former president of Ukraine, Victor Yushchenko. These persistent organic pollutants are by-products of industrial activity and bind to an intracellular receptor, AhR, with a high potency. In humans, exposure to dioxins, in particular 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a cutaneous syndrome known as chloracne, consisting in the development of many small skin lesions (hamartoma), lasting for 2–5 years. Although TCDD has been classified by the WHO as a human car-cinogen, its carcinogenic potential to humans is not clearly demonstrated. It was first believed that AhR activation accounted for most, if not all, biological properties of dioxins. However, certain AhR agonists found in vegetables do not induce chloracne, and other chemicals, in particular certain therapeutic agents, may induce a chloracne-like syndrome without activating AhR. It is time to rethink the mechanism of dioxin toxicity and analyse in more details the biological events following exposure to these compounds and other AhR agonists, some of which have a very different chemical structure than TCDD. In particular various food-containing AhR agonists are non-toxic and may on the contrary have beneficial properties to human health. © 2013 Elsevier Ireland Ltd. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 2. AhR signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 17
  18. 18. Effets aigüs Le cas Iouchtchenko 2000 2004 Empoisonnement au TCDD de Viktor Iouchtchenko (alors premier ministre ukrainien) (6 sept 2004) 18
  19. 19. TOXICOLOGICAL SCIENCES 125(1), 310–317 (2012) doi:10.1093/toxsci/kfr223 Advance Access publication October 13, 2011 19 The Cutaneous Lesions of Dioxin Exposure: Lessons from the Poisoning of Victor Yushchenko Jean-Hilaire Saurat,*,†,1 Guerkan Kaya,*,† Nikolina Saxer-Sekulic,*,† Bruno Pardo,* Minerva Becker,‡ Lionel Fontao,† Florence Mottu,*,† Pierre Carraux,† Xuan-Cuong Pham,† Caroline Barde,† Fabienne Fontao,* Markus Zennegg,§ Peter Schmid,§ Olivier Schaad,{ Patrick Descombes,{ and Olivier Sorg*,† *Swiss Centre for Applied Human Toxicology, Dermatotoxicology Unit, University of Geneva, 1211 Geneva 4, Switzerland; †Dermatology Department and ‡Radiology Department, Geneva University Hospital, 1211 Geneva 14, Switzerland; §EMPA (Swiss Federal Laboratories for Materials Testing and Research), 8600 Du¨bendorf, Switzerland; and {Genomics Platform, National Center of Competence in Research Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland 1To whom correspondence should be addressed at Swiss Centre for Applied Human Toxicology, University of Geneva, 1, rue Michel-Servet, 1211 Gene`ve 4, Switzerland. Fax: 0041-22-379 5502. E-mail: jean.saurat@unige.ch. Received August 10, 2011; accepted August 10, 2011 Several million people are exposed to dioxin and dioxin-like characterization of dioxin exposure remains difficult to establish, … pour la Science
  20. 20. of sampling were prepared as previously reported (Sorg et al., 2008). RNA TOXICOLOGICAL SCIENCES 125(1), 310–317 (2012) quality doi:10.1093/toxsci/was kfr223 assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Advance Access publication October 13, 2011 Nano LabChip kit. We generated a hybridization mixture containing 15 lg of biotinylated complementary RNA and hybridized it to GeneChip HG U133 Plus 2.0 according to manufacturer’s instructions (Affymetrix). To identify differentially expressed transcripts, comparisons were carried out after normalization with the Affymetrix GCOS 1.2 (MAS5) software. The Cutaneous Lesions of Dioxin Exposure: Lessons from the Poisoning of Victor Yushchenko Jean-Hilaire Saurat,*,†,1 Guerkan Kaya,*,† Nikolina Saxer-Sekulic,*,† Bruno Pardo,* Minerva Becker,‡ Lionel Fontao,† Florence Mottu,*,† Pierre Carraux,† Xuan-Cuong Pham,† Caroline Barde,† Fabienne Fontao,* Markus Zennegg,§ Bioinformatic analysis. Responsive elements for genes corresponding to Peter Schmid,§ Olivier Schaad,{ Patrick Descombes,{ and Olivier Sorg*,† differentially expressed transcripts were searched with the University of small fiber peripheral neuropathy (The organs and systems involved, other than the skin, are just cited here. Each will be fully addressed in future publications when the mechanism has been better analyzed by appropriate ongoing data analysis.). Facial involvement worsened with diffuse nodular lesions on an edematous background, sparing of the periocular zone, but major involvement of the ears and retroauricular folds (Figs. 2A and 2B). The basic skin lesions were small nodules (Fig. 2C), *Swiss Centre for Applied Human Toxicology, Dermatotoxicology Unit, University of Geneva, 1211 Geneva 4, Switzerland; †Dermatology Department and ‡Radiology Department, Geneva University Hospital, 1211 Geneva 14, Switzerland; §EMPA (Swiss Federal Laboratories for Materials Testing and Research), 8600 Du¨bendorf, Switzerland; and {Genomics Platform, National Center of Competence in Research Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland 1To whom correspondence should be addressed at Swiss Centre for Applied Human Toxicology, University of Geneva, 1, rue Michel-Servet, 1211 Gene`ve 4, Switzerland. Fax: 0041-22-379 5502. E-mail: jean.saurat@unige.ch. Received August 10, 2011; accepted August 10, 2011 Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called ‘‘chloracne’’ are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics. Key Words: dioxin; toxicity; skin; hamartoma; morphology. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is the most potent of a large number of industrial-era halogenated polyaromatic hydrocarbon pollutants, including other dibenzo-p-dioxins, dibenzofurans, and certain polychlorinated biphenyls. Human populations are exposed to low levels of dioxin and dioxin-like compounds, primarily through food consumption (Connor et al., 2008; Schecter et al., 1999). The risk characterization of dioxin exposure remains difficult to establish, although it is an issue that broadly affects important public health policy decisions (Gies et al., 2007; Steenland et al., 2001). Thus, chronic exposure to low/moderate doses of dioxin may be involved not only in the classic dioxin toxicity in some genetically predisposed individuals (IARC Monograph, 1997; Aylward et al., 2005) but also in the newly identified role of these compounds in autoimmunity (Brembilla et al., 2011; Marshall and Kerkvliet, 2010; Ramirez et al., 2010). In humans, skin lesions called ‘‘chloracne’’ are the most visible and consistent response to dioxin exposure and therefore play the role of a sentinel sign for toxicity (Caputo et al., 1988). The mechanism by which chloracne appears was not previously known and its diagnostic value is not straightforward, especially in mild and sporadic cases, which could still relate to significant exposure (Passarini et al., 2010). A robust indicator that would trigger specific ecotoxicology diagnostic processes is lacking; in the current situation, it is likely that many cases have not been recognized (Saurat and Sorg, 2010). We have previously reported on the TCDD poisoning in Victor Yushchenko with identification and measurement of TCDD metabolites (Sorg et al., 2009). The maximum accepted daily dose exposure in human is 4 pg/kg, and this patient received a single dose of 20 lg/kg. With the approval of the patient to release peer-reviewed scientific information on his case, we now report on a set of data that has never been obtained in humans and helps define the phenotype of the dioxin-induced skin pathology. FIG. 1. Evolution of the dioxin disease. Chronology of organ involvement a.p. The peak of skin involvement is delayed as compared with the other organs, and skin lesions show a longer and chronic course. MATERIALS AND METHODS Clinical specimens. Skin sampling was performed under general anesthe-sia during therapeutic procedures. ! The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from http://toxsci.oxfordjournals.org/ by guest on October 1, 2014 20
  21. 21. SCIENCES 125(1), 310–317 (2012) Downloaded TOXICOLOGICAL doi:10.1093/toxsci/kfr223 Advance Access publication October 13, 2011 The Cutaneous Lesions of Dioxin Exposure: Lessons from the Poisoning FROM THE POISONING OF VICTOR YUSHCHENKO 315 sebum lipid ! Downloaded the promoter and sterol O-acyltransferase tissue renewing induced genes, factor A serine of the bone highly relevant to al., 2006). from structural/ extracellular matrix’’ expected from specific pattern. strongly repressed involved in the of Victor Yushchenko Jean-Hilaire Saurat,*,†,1 Guerkan Kaya,*,† Nikolina Saxer-Sekulic,*,† Bruno Pardo,* Minerva Becker,‡ Lionel Fontao,† Florence Mottu,*,† Pierre Carraux,† Xuan-Cuong Pham,† Caroline Barde,† Fabienne Fontao,* Markus Zennegg,§ Peter Schmid,§ Olivier Schaad,{ Patrick Descombes,{ and Olivier Sorg*,† *Swiss Centre for Applied Human Toxicology, Dermatotoxicology Unit, University of Geneva, 1211 Geneva 4, Switzerland; †Dermatology Department and ‡Radiology Department, Geneva University Hospital, 1211 Geneva 14, Switzerland; §EMPA (Swiss Federal Laboratories for Materials Testing and Research), 8600 Du¨bendorf, Switzerland; and {Genomics Platform, National Center of Competence in Research Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland 1To whom correspondence should be addressed at Swiss Centre for Applied Human Toxicology, University of Geneva, 1, rue Michel-Servet, 1211 Gene`ve 4, Switzerland. Fax: 0041-22-379 5502. E-mail: jean.saurat@unige.ch. Received August 10, 2011; accepted August 10, 2011 Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called ‘‘chloracne’’ are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics. Key Words: dioxin; toxicity; skin; hamartoma; morphology. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is the most potent of a large number of industrial-era halogenated polyaromatic hydrocarbon pollutants, including other dibenzo-p-dioxins, dibenzofurans, and certain polychlorinated biphenyls. Human populations are exposed to low levels of dioxin and dioxin-like compounds, primarily through food consumption (Connor et al., 2008; Schecter et al., 1999). The risk characterization of dioxin exposure remains difficult to establish, although it is an issue that broadly affects important public health policy decisions (Gies et al., 2007; Steenland et al., 2001). Thus, chronic exposure to low/moderate doses of dioxin may be involved not only in the classic dioxin toxicity in some genetically predisposed individuals (IARC Monograph, 1997; Aylward et al., 2005) but also in the newly identified role of these compounds in autoimmunity (Brembilla et al., 2011; Marshall and Kerkvliet, 2010; Ramirez et al., 2010). In humans, skin lesions called ‘‘chloracne’’ are the most visible and consistent response to dioxin exposure and therefore play the role of a sentinel sign for toxicity (Caputo et al., 1988). The mechanism by which chloracne appears was not previously known and its diagnostic value is not straightforward, especially in mild and sporadic cases, which could still relate to significant exposure (Passarini et al., 2010). A robust indicator that would trigger specific ecotoxicology diagnostic processes is lacking; in the current situation, it is likely that many cases have not been recognized (Saurat and Sorg, 2010). We have previously reported on the TCDD poisoning in Victor Yushchenko with identification and measurement of TCDD metabolites (Sorg et al., 2009). The maximum accepted daily dose exposure in human is 4 pg/kg, and this patient received a single dose of 20 lg/kg. With the approval of the patient to release peer-reviewed scientific information on his case, we now report on a set of data that has never been obtained in humans and helps define the phenotype of the dioxin-induced skin pathology. MATERIALS AND METHODS Clinical specimens. Skin sampling was performed under general anesthe-sia during therapeutic procedures. from http://toxsci.oxfordjournals.org/ by guest on October 1, 2014 21
  22. 22. Compassionate use of tumor necrosis factor a (TNF-a) blockade was considered because non-steroidal anti-inflammatory drugs and systemic steroids were not effective. The patient received three infusions of infliximab but because of intolerancewas then switched to adalimumab, which was given for M18 (M16th to M34th a.p.). AL. ! Downloaded FIG. 5. Volumetric analyses of the skin lesions. (A) Three-dimensional representation of the calculated total skin volume of the face using the methodology described in the text. (B) Three-dimensional representation of the calculated volume of the hamartomatous lesions seen in the same area as in (A). Anterior view. Note that most lesions are located in the ear lobes and lateral cheeks. (C) Thick slab reconstruction of an FDG PET data set obtained from a whole-body PET/CT acquisition showing the distribution and the metabolism org/ by guest on October 1, 2014 TOXICOLOGICAL SCIENCES 125(1), 310–317 (2012) doi:10.1093/toxsci/kfr223 Advance Access publication October 13, 2011 The Cutaneous Lesions of Dioxin Exposure: Lessons from the Poisoning of Victor Yushchenko Jean-Hilaire Saurat,*,†,1 Guerkan Kaya,*,† Nikolina Saxer-Sekulic,*,† Bruno Pardo,* Minerva Becker,‡ Lionel Fontao,† Florence Mottu,*,† Pierre Carraux,† Xuan-Cuong Pham,† Caroline Barde,† Fabienne Fontao,* Markus Zennegg,§ Peter Schmid,§ Olivier Schaad,{ Patrick Descombes,{ and Olivier Sorg*,† *Swiss Centre for Applied Human Toxicology, Dermatotoxicology Unit, University of Geneva, 1211 Geneva 4, Switzerland; †Dermatology Department and ‡Radiology Department, Geneva University Hospital, 1211 Geneva 14, Switzerland; §EMPA (Swiss Federal Laboratories for Materials Testing and Research), 8600 Du¨bendorf, Switzerland; and {Genomics Platform, National Center of Competence in Research Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland 1To whom correspondence should be addressed at Swiss Centre for Applied Human Toxicology, University of Geneva, 1, rue Michel-Servet, 1211 Gene`ve 4, Switzerland. Fax: 0041-22-379 5502. E-mail: jean.saurat@unige.ch. Received August 10, 2011; accepted August 10, 2011 Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called ‘‘chloracne’’ are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics. Key Words: dioxin; toxicity; skin; hamartoma; morphology. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is the most potent of a large number of industrial-era halogenated polyaromatic hydrocarbon pollutants, including other dibenzo-p-dioxins, dibenzofurans, and certain polychlorinated biphenyls. Human populations are exposed to low levels of dioxin and dioxin-like compounds, primarily through food consumption (Connor et al., 2008; Schecter et al., 1999). The risk characterization of dioxin exposure remains difficult to establish, although it is an issue that broadly affects important public health policy decisions (Gies et al., 2007; Steenland et al., 2001). Thus, chronic exposure to low/moderate doses of dioxin may be involved not only in the classic dioxin toxicity in some genetically predisposed individuals (IARC Monograph, 1997; Aylward et al., 2005) but also in the newly identified role of these compounds in autoimmunity (Brembilla et al., 2011; Marshall and Kerkvliet, 2010; Ramirez et al., 2010). In humans, skin lesions called ‘‘chloracne’’ are the most visible and consistent response to dioxin exposure and therefore play the role of a sentinel sign for toxicity (Caputo et al., 1988). The mechanism by which chloracne appears was not previously known and its diagnostic value is not straightforward, especially in mild and sporadic cases, which could still relate to significant exposure (Passarini et al., 2010). A robust indicator that would trigger specific ecotoxicology diagnostic processes is lacking; in the current situation, it is likely that many cases have not been recognized (Saurat and Sorg, 2010). We have previously reported on the TCDD poisoning in Victor Yushchenko with identification and measurement of TCDD metabolites (Sorg et al., 2009). The maximum accepted daily dose exposure in human is 4 pg/kg, and this patient received a single dose of 20 lg/kg. With the approval of the patient to release peer-reviewed scientific information on his case, we now report on a set of data that has never been obtained in humans and helps define the phenotype of the dioxin-induced skin pathology. MATERIALS AND METHODS Clinical specimens. Skin sampling was performed under general anesthe-sia during therapeutic procedures. from http://toxsci.oxfordjournals.org/ by guest on October 1, 2014 22 FIG. 3. Histological analyses of the skin lesions. (A) Photomicrograph of
  23. 23. Prélèvements et analyses PCB et composés dioxin-like 23
  24. 24. 24 Ordonnance sur les travaux de construction Obligation de planification des travaux si la présence de PCB est suspectée
  25. 25. fixe une limite de teneur de PCB dans les joints de 50 ppm (mg/kg) Appliqué par extension aux autres matériaux (peintures) 25
  26. 26. 26
  27. 27. Analyse des PCBs 27
  28. 28. Les PCB indicateurs les 6 grands électeurs PCB 28 PCB 101 PCB 153 PCB 52 PCB 138 PCB 180 28
  29. 29. 1016$ 1242$ 1248$ 1248$ 1254$"Late"$ 1254$ 1260$ 0$ 2$ 4$ 6$ 8$ 10$ 12$ 14$ 1$ 4$ 7$ 10$ 13$ 16$ 19$ 22$ 25$ 28$ 31$ 34$ 37$ 40$ 43$ 46$ 49$ 52$ 55$ 58$ 61$ 64$ 67$ 70$ 73$ 76$ 79$ 82$ 85$ 88$ 91$ 94$ 97$ 100$ 103$ 106$ 109$ 112$ 115$ 118$ 121$ 124$ 127$ 130$ 133$ 136$ 139$ 142$ 145$ 148$ 151$ 154$ 157$ 160$ 163$ 166$ 169$ 172$ 175$ 178$ 181$ 184$ 187$ 190$ 193$ 196$ 199$ 202$ 205$ 208$ %"masse" 1016$ 1242$ 1248$ 1248$ 1254$"Late"$ 1254$ 1260$ La composition des Aroclors 29 Données sources : ATDSR
  30. 30. Les facteurs de conversion 30 version massique 28 52 101 138 153 180 Total % Facteur 1016 8.50 4.63 0.04 13.2 7.6 1242 6.86 3.53 0.69 0.10 0.06 11.2 8.9 1248 3.59 6.93 2.22 0.38 0.23 0.02 13.4 7.5 1248 5.57 5.58 1.89 0.41 0.43 0.21 14.1 7.1 1254 "Late" 0.06 0.83 5.49 5.95 3.29 0.42 16.0 6.2 1254 0.19 5.38 8.02 5.80 3.77 0.67 23.8 4.2 1260 0.03 0.24 3.13 6.54 9.39 11.38 30.7 3.3 Aroclor PCB
  31. 31. Et au niveau toxicité des 31 PCB-dl ? 77 0.0001 81 0.0003 126 0.1 169 0.03 105 0.00003 114 0.00003 118 0.00003 123 0.00003 156 0.00003 157 0.00003 167 0.00003 189 0.00003 %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq %masse Teq 1016 1242 0.31 3E-05 0.01 3E-06 0.47 1E-05 0.04 1E-06 0.66 2E-05 0.03 9E-07 0.01 3E-07 1248 0.41 4E-05 0.01 3E-06 1.6 5E-05 0.12 4E-06 2.29 7E-05 0.07 2E-06 0.06 2E-06 0.01 3E-07 0.01 3E-07 1248 0.52 5E-05 0.02 6E-06 1.45 4E-05 0.12 4E-06 2.35 7E-05 0.08 2E-06 0.04 1E-06 0.01 3E-07 1254 "Late" 0.2 2E-05 0.02 0.002 7.37 0.0002 0.5 2E-05 13.59 4E-04 0.32 1E-05 1.13 3E-05 0.3 9E-06 0.35 1E-05 0.01 3E-07 1254 0.03 3E-06 2.99 9E-05 0.18 5E-06 7.35 2E-04 0.15 5E-06 0.82 2E-05 0.19 6E-06 0.27 8E-06 0.01 3E-07 1260 0.22 7E-06 0.48 1E-05 0.52 2E-05 0.02 6E-07 0.19 6E-06 0.1 3E-06 Teq (ng/g) pour 50 ppm 28 52 101 138 153 180 Total % Facteur 1016 8.50 4.63 0.04 13.2 7.6 1242 6.86 3.53 0.69 0.10 0.06 11.2 8.9 1248 3.59 6.93 2.22 0.38 0.23 0.02 13.4 7.5 1248 5.57 5.58 1.89 0.41 0.43 0.21 14.1 7.1 1254 "Late" 0.06 0.83 5.49 5.95 3.29 0.42 16.0 6.2 1254 0.19 5.38 8.02 5.80 3.77 0.67 23.8 4.2 1260 0.03 0.24 3.13 6.54 9.39 11.38 30.7 3.3 Aroclor PCB Teq$ng/kg 1016 0 1242 5 1248 39 1248 41 1254 "Late" 3244 1254 217 1260 4
  32. 32. OK, les PCB indicateurs c’est pourri, mais comment on peut mesurer un Teq TCDD ? La question que le premier rang se pose
  33. 33. GC-HRMS 33 Profil GC-HRMS d’un extrait de cendres volantes. © Restek
  34. 34. pg eq TCDD / gr échantillon 34 Oui, mais 7 PCDD sur 75 10 PCDF sur 135 12 PBC-dl sur 12 … et les autres ligands ? … et les synergies ?
  35. 35. Au secours DR CALUX !!! Dr Heinrich von Calux 1875 - 1910 Peut-on mesure35r réellement un Teq TCDD alors ?
  36. 36. Dioxin Receptor Chemically Activated LUciferase gene eXpression 36 Vous allez voir, c’est tout simple Méthode de biologie moléculaire de mesure de l’induction d’un récepteur par un ligand.
  37. 37. 37 Voyez, c’est simple
  38. 38. 38 NOYAU Moins simple
  39. 39. 39 … désolé
  40. 40. Principe du gène rapporteur (reporteur gene) 40
  41. 41. DR-CALUX méthodologie 41
  42. 42. GC-HRMS vs DR-CALUX 42 GC-HRMS DR-CALUX Spécificité +++ Substances + Capacité d’activation du récepteur Sensibilité ++ 1 pg eq TCDD +++ 0.3 pg eq TCDD Capacité de screening ++ Analyse par substance +++ Détection des ligands inconnus Détection des effets de synergie (coktail) Mise en oeuvre Coût : env 1000 CHF Rapidité : env 15 jours Coût : env 250 CHF Rapidité : env 5 jours Validation Golden Standard Validations, food, sang, terres, poussières, eaux.
  43. 43. Exemples d’application du DR CALUX en hygiène du travail Suivi des expositions des travailleurs lors des travaux de sécurisation d’un site contaminé aux PCB. 43
  44. 44. Mesure de la perméation des PCB indicateurs dans les combinaisons
  45. 45. Suivi sanguin DR-CALUX analyse Teq TCDD sang A (avant travaux): 21.8 +/- 5.03 pg TCDD TEQ/g fat B: (après travaux): 19.9 +/- 6.42 pg TCDD TEQ/g fat Pas de différence statistiquement significative entre les 2 séries (Wilcoxon signed rank test, paired values) No patient 1 2 3 4 5 6 7 8 9 10 Average Median Standard deviation Coeff. of variation Minimum Maximum Range Stnd. skewness Stnd. kurtosis CALUX A PCDD/PCDF and dl- PCBs (only total TEQ) [pg TEQ/g fat] CALUX B PCDD/PCDF and dl- PCBs (only total TEQ) [pg TEQ/g fat] 19* 29 28 25 32 18 20 16 24 16 20 13 16* 17 23 21 19 31 17 13 21.8 19.9 20 17.5 5.03 6.42 0.23 0.32 16 13 32 31 16 18 1.34 0.98 0.31 -0.50 (*) < LOQ Concentrations usuelles dans le sérum (publications) Non exposé ou nouveau né: 23 to 27 pg TCDD TEQ/g fat Habitant proche d’une usine d’incinération 55 to 76 pg TCDD TEQ/g fat T0 T+4 mois 45
  46. 46. Exemples d’application du DR CALUX en environnement Analyse des PCB dans des éléments en amiante-ciment 46
  47. 47. Concentration*TCDD-TEQ Etat pg/g*TEQ pg/cm2*TEQ Site*A*-*gris*clair <*0.067 <*0.12 Site*A*-*gris*foncé*après*incendie 1.900 0.50 Site*B*-*peinture*de*surface*rouge 0.940 2.70 Site*C*-*gris*clair 0.400 0.34 Site*D*-*gris*foncé 0.066 0.07 Site*E*-*gris*foncé 0.037 0.05 Site*F*-*gris*clair 0.029 0.03 Site*G*-*gris*clair 0.061 0.05 47 Ech.%2 Ech.%3 PCB$indicateurs pg/g pg/g PCB'28 <5.8 <0.24 PCB'52 <5.8 14 PCB'101 28 58 PCB'138 103 57 PCB'153 71 46 PCB'180 141 13 Somme'des'7'PCB'indicateurs 344 188 1.72 ppm < 50 ppm Ech.%2 Ech.%3 PCB$dioxin$like pg/g pg/g PCB'77 <5.8 11 PCB'105 81 29 PCB'114 <5.8 <0.24 PCB'118 49 30 PCB'123 <5.8 <0.24 PCB'126 <5.8 1.6 PCB'156 <5.8 8.3 PCB'157 <5.8 2.6 PCB'167 <5.8 3.9 PCB'169 <5.8 <0.24 PCB'189 <5.8 <0.24 WHO(2005)'PCB'TEQ 2.5 0.19 8 échantillons d’éléments en amiante-ciment (Suisse Romande) TOXpro 2014, unpublished data
  48. 48. Toiture : 13 pg/g PCB-dl (Teq TCDD) Prélèvement de surface 345’141 pg/g PCB-dl (Teq TCDD) Sol 139 pg/g PCD-dl (Teq TCDD) 48
  49. 49. Exemples d’application du DR CALUX en hygiène du travail Contamination des surfaces de travail aux composés Dioxin-like dans un incinérateur municipal
  50. 50. 50 Prélèvement des surfaces (wipe-test) TOXpro 2014, unpublished data Valeurs seuils Allemagne 10 ng/m2
  51. 51. SIG$D13$($fines$sous$grille$ 2378*TetraCDD# 40%# 30%# 20%# 10%# 0%# 12378*PentaCDD# 123478*HexaCDD# 123678*HexaCDD# 123789*HexaCDD# 1234678*HeptaCDD# 12346789*OctaCDD# 2378*TetraCDF# 12378*PentaCDF# 23478*PentaCDF# 123478*HexaCDF# 123678*HexaCDF# 123789*HexaCDF# 234678*HexaCDF# 12346789*OctaCDF# 1234789*HeptaCDF# 1234678*HeptaCDF# PCB#77# PCB#81# PCB#123# PCB#118# PCB#114# PCB#169# PCB#126# PCB#105# PCB#156# PCB#157# PCB#167# PCB#189# PCDD$ PCDF$ PCB'dl$ SIG$D3$'$Cendres$volantes$ 2378*TetraCDD# 40%# 30%# 20%# 10%# 0%# 12378*PentaCDD# 123478*HexaCDD# 123678*HexaCDD# 123789*HexaCDD# 1234678*HeptaCDD# 12346789*OctaCDD# 2378*TetraCDF# 12378*PentaCDF# 23478*PentaCDF# 123478*HexaCDF# 123678*HexaCDF# 123789*HexaCDF# 234678*HexaCDF# 12346789*OctaCDF# 1234789*HeptaCDF# 1234678*HeptaCDF# PCB#77# PCB#81# PCB#123# PCB#118# PCB#114# PCB#169# PCB#126# PCB#105# PCB#156# PCB#157# PCB#167# PCB#189# PCDD$ PCDF$ PCB'dl$ 51 Recherche des sources par GC-HRMS Cendres volantes Dépôt sur armoire TOXpro 2014, unpublished data
  52. 52. Mais bon, y’a pas que les PCB / Dioxines dans la vie ! PCB et composés dioxin-like
  53. 53. Les retardateurs de flamme Les retardateurs de flamme 53
  54. 54. 54 Brommer et al. J Environ Monitor 2012; Marklund et al. Chemosphere 2003; Stapleton et al. Environ Sci Technol 2005;2009; Takigami et al. Environ Int 2009; Van den Eede et al. Environ Int 2011
  55. 55. profiles to in vivo toxicity of the compounds? La gamme des récepteurs et « pathway) 55 compound ‘profile’ Compound CALUX assays currently available: Nuclear receptors Signaling pathways name endpoint name endpoint DR CALUX dioxins NFκB CALUX inflammation PAH CALUX PAHs p21 CALUX DNA damage ERα CALUX estrogens Nrf2 CALUX oxid. stress ERβ CALUX estrogens p53 CALUX DNA damage AR CALUX androgens TCF CALUX carcinogenesis PR CALUX progestins AP1 CALUX stress GR CALUX glucocortocoid HIF1α CALUX hypoxia TRβ CALUX thyroids ESRE CALUX ER stress RAR CALUX retinoids Cytotox CALUX cytotoxicity PPARγ CALUX obesogens PPARα CALUX obesogens PPARδ CALUX obesogens PXR CALUX xenobiotics LXR CALUX oxysterols
  56. 56. Les possibilités de screening 56
  57. 57. Screening des micropolluants, eaux uséés 57
  58. 58. OUF ! 58

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