dioxines, pcb, furanes et dr-calux

Exposition aux PCB et Dioxines, qu’en pense le Dr CALUX ?

Vincent PERRET Hygiéniste du travail certifié SSHT

Groupement romand de médecine, d’hygiène et de sécurité au travail Journée de présentation de cas 02 octobre 2014

Hygiène du travail Toxicologie industrielle

Les PCB quelques points

PolyChloroBiphenyles

209 congénères

•  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)

2

Nomenclature des PCB Nomenclature de Ballschmiter & Zell

3

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

Exemples d’application des PCB

5


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


Peintures industrielles (1955-1975) < 20’000 mg/kg (ppm)

7


Peintures ignifuge de faux-plafonds

8


Eléments bitumes d’étanchéité de toiture Source EPA

9

Eléments en amiante ciment Nombreux cas de contamination d’œufs aux PCBs dans des fermes du nord de la Hollande et d’Allemagne


10

Effets sur la santé PCB et composés dioxin-like

11

Polychlorodibenzodioxines (PCDD) 75 congénères

Polychlorodibenzofuranes (PCDF) 135 congénères

Polychlorobiphényls (PCB) 209 congénères

Les PCB baby dioxine ?

12 congénères planaires

12

Toxicité relative des composés dioxines, furanes et PCB-dl

Facteurs d’équivalence toxique relatif au 2,3,7,8- TCDD

13

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

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

Hamster Golden Syrian

Cochon d’inde Hartley

Face à la dioxine, qui est le plus fort ?

16

O. Sorg / Toxicology Letters 230 (2014) 225–233 229

Table 2Relation between TCDD LD50 and body fat precentage.

Species (strain) Body fat [%] LD50 [!g/kg]

Guinea pig (Hartley) 4.5 1American dark mink 4Hare 7.5 10Chicken 35Macaque 10 50Rat (Sprague-Dawley) 10 50Dog 100Rabbit 10 120Moiuse (C57BL) 8 150Rat (Fischer) 10 300Mouse (BALB/c) 400Dog (Beagle) 14 1000Frog 1000Mouse (DBA) 20 2500Hamster (golden Syrian) 15 10,000

one of the most resistant species with a LD50 of ≈12.5 mg/kg! How-ever, the patient we examined in the years 2005 to 2009 developedlife-threatening conditions such as toxic pancreatitis and hepatitisshortly after receiving an estimated TCDD dose of 20 !g/kg (Sorget al., 2009), indicating that the human LD50 for TCDD might besignificantly lower than previously estimated.

Following acute TCDD intoxication, the target organs developa pathology and recover with very different kinetics: the GI tract,the liver and the pancreas are the first organs to be affected andrecover within 6 to 10 weeks, whereas the clinical manifestationsof the skin start to develop only after several weeks, reach a peak at18 months and decrease slowly over a period of 3 to 5 years (Sauratet al., 2012).

4.2. The skin as a target of dioxin toxicity

The hallmark of the clinical manifestations of the skin followingdioxin exposure is the development of numerous dermal hamar-toma over a long period of time (1–3 years). When analysingin detail these lesions in a patient exposed to TCDD we calledthem MADISH, for “metabolising acquired dioxin-induced skinhamartoma” (Saurat et al., 2012; Saurat and Sorg, 2010). The mor-phological analysis of the affected skin revealed the disappearanceof sebaceous glands. The kinetics of “MADISH” development, theirdensity and the concomitant sebaceous gland atrophy led us tohypothesize that the cutaneous stem cells that normally differen-tiate to sebocytes to maintain sebaceous gland turnover undergoa genetic switch by dioxin and produce these “MADISH” insteadof new sebocytes. Indeed the main enzymes involved in specificlipids found in sebum are highly repressed following acute dioxinexposure (Saurat et al., 2012).

Fig. 4. TCDD oral LD50 vs. body fat percentage. See text for details (dioxin toxic-ity/acute dioxin toxicity). Adapted from Geyer et al. (1993).

4.3. Controversy about dioxin carcinogenicity

Although TCDD is classified as a human carcinogen by the WHOand the US National Toxicological Program, there is a strong matterof debate regarding the carcinogen potential of TCDD to humans.A completed report by the IARC on the evaluation of the carcino-genicity of PCDD and PCDF in humans, published at the time whereTCDD was considered by WHO as a possible human carcinogen,was not conclusive (IARC, 1997). This classification results morefrom an application of the precaution principle than retrospectiveclinical trials in humans. Evidence for TCDD as a carcinogen camefrom (1) some animal and theoretical models (Kohn, 1995; Polandet al., 1982; Simon et al., 2009), (2) mutagenic metabolic conver-sions of other polycyclic aromatic hydrocarbons (Audebert et al.,2010; Brookes, 1977; Mastrangelo et al., 1996) and (3) relation-ships between AhR signalling and expression of genes involved inoncogenesis (Seifert et al., 2009; Villano et al., 2006). However,

a) owing to the great variety of sensitivity of animals to TCDD (e.g.the Syrian hamster is 10,000-fold less sensitive than the Guineapig), it is difficult to extrapolate animal data regarding TCDDbiology directly to humans (Scheuplein and Bowers, 1995);

b) a metabolic mutagenic conversion of a polycyclic aromatichydrocarbon happens when the inducible CYP1A1 activity con-verts the parent hydrocarbon to a relatively stable epoxide, thelatter being mutagenic (Brookes, 1977; Mastrangelo et al., 1996).In the case of TCDD, although CYP1A1 is highly induced, themetabolism is so slow that the biological half-life of TCDD is7-10 years. The rate of epoxide formation is thus so insignificantthat it has no biological consequences;

c) Many genes may be modulated by TCDD, as demonstrated bythe whole genome transcriptome of V. Yushchenko (Sauratet al., 2012), so the biological interpretation is not straightfor-ward. Moreover, AhR signalling pathway may induce cross-talkswith other signalling pathways, rendering the interpretationof the modulation of some genes by AhR activation very diffi-cult. In particular, AhR activation increases the activity of thetranscription factor Nrf2, associated to cytoprotection againstdegenerative diseases (Hayes et al., 2009).

d) All retrospective clinical trials on cancer prevalence in the pop-ulation of Seveso exposed in 1976 to TCDD fail to demonstrate acausal link between TCDD exposure and cancer (Bertazzi et al.,2001; Boffetta et al., 2011).

As a conclusion, to date we have no evidence to state that TCDDis a significant carcinogen in humans (Cole et al., 2003; Tuomistoand Tuomisto, 2012).

4.4. Reprotoxicity of dioxin

Retrospective studies following the accident in Seveso (North-ern Italy) in 1976 demonstrated a decreased male/female sex ratioin children born to males exposed to TCDD (Mocarelli et al., 2000),as well as an endocrine disrupting activity affecting semen qual-ity in young males (Mocarelli et al., 2008). These observations andthe fact that AhR activation may induce the estrogen signallingpathways make TCDD a possible endocrine disruptor. This wasconfirmed by various animal studies showing the suppression ofplacental vascular remodelling in rats (Ishimura et al., 2006), a lossof fertilized oocytes in mice (Kitajima et al., 2004) and an inhibitionof follicular development in zebrafish (Heiden et al., 2006). How-ever, studies in humans are less conclusive. A retrospective studyon American workers, who were exposed to TCDD during the pro-duction of the Agent Orange herbicide, failed to find a causative linkbetween TCDD exposure of males and a rate of spontaneous abor-tion in their wives (Schnorr et al., 2001). It is clear that the level of

Toxicology Letters 230 (2014) 225–233

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

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

h i g h l i g h t s

• Besides its canonical pathway,AhR may activate other receptor-mediated pathways.

• AhR activity may be assessed bychemical or biological assays.

• Dioxin toxicity cannot be explainedonly by AhR activation.

• AhR activation leads to either upre-gulation or downregulation of genes.

• TCDD as a human carcinogen is still amatter of debate.

• Natural AhR agonists 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 2013Received in revised form 14 October 2013Accepted 18 October 2013Available online 12 November 2013

Keywords:DioxinTCDDAhRCell signallingSkinToxicity

a b s t r a c t

Dioxins are a family of molecules associated to several industrial accidents such as Ludwigshafen in1953 or Seveso in 1976, to the Agent Orange used during the war of Vietnam, and more recently to thepoisoning of the former president of Ukraine, Victor Yushchenko. These persistent organic pollutantsare 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 acutaneous 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 AhRactivation accounted for most, if not all, biological properties of dioxins. However, certain AhR agonistsfound 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 ofdioxin toxicity and analyse in more details the biological events following exposure to these compoundsand other AhR agonists, some of which have a very different chemical structure than TCDD. In particularvarious food-containing AhR agonists are non-toxic and may on the contrary have beneficial propertiesto human health.

© 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2262. AhR signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Abbreviations: AhR, aromatic hydrocarbon receptor; MADISH, metabolising acquired dioxin-induced skin hamartoma; NAHRA, natural AhR agonist; PCB, polychlorinatedbiphenyls; PCDD, polychlorinated dibenzodioxins; PCDF, polychlorinated dibenzofurans; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEF, TCDD-equivalent factor; TEQ,TCDD-equivalent.

∗ Corresponding author. Tel.: +41 22 3795032.E-mail address: [email protected]

0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.toxlet.2013.10.039

17

Effets aigüs Le cas Iouchtchenko

2000 2004 Empoisonnement au TCDD de Viktor Iouchtchenko (alors premier ministre ukrainien) (6 sept 2004)

18

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 Poisoningof 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 Dubendorf, Switzerland; and {Genomics Platform, National Center of Competence in Research Frontiers in Genetics, University of Geneva, 1211Geneva 4, Switzerland

1To whom correspondence should be addressed at Swiss Centre for Applied Human Toxicology, University of Geneva, 1, rue Michel-Servet, 1211 Geneve 4,

Switzerland. Fax: 0041-22-379 5502. E-mail: [email protected].

Received August 10, 2011; accepted August 10, 2011

Several million people are exposed to dioxin and dioxin-likecompounds, primarily through food consumption. Skin lesionshistorically called ‘‘chloracne’’ are the most specific sign ofabnormal dioxin exposure and classically used as a key marker inhumans. We followed for 5 years a man who had been exposed tothe most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD), at a single oral dose of 5 million-fold more than theaccepted daily exposure in the general population. We adopteda molecular medicine approach, aimed at identifying appropriatetherapy. Skin lesions, which progressively covered up to 40% ofthe body surface, were found to be hamartomas, which developedparallel to a complete and sustained involution of sebaceousglands, with concurrent transcriptomic alterations pointing to theinhibition of lipid metabolism and the involvement of bonemorphogenetic proteins signaling. Hamartomas created a newcompartment that concentrated TCDD up to 10-fold comparedwith serum and strongly expressed the TCDD-metabolizingenzyme cytochrome P450 1A1, thus representing a potentiallysignificant source of enzymatic activity, which may add to thexenobiotic metabolism potential of the classical organs such as theliver. This historical case provides a unique set of data on thehuman tissue response to dioxin for the identification of newmarkers of exposure in human populations. The herein discoveredadaptive cutaneous response to TCDD also points to the potentialrole 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 themost potent of a large number of industrial-era halogenatedpolyaromatic hydrocarbon pollutants, including other dibenzo-p-dioxins, dibenzofurans, and certain polychlorinated biphenyls.

Human populations are exposed to low levels of dioxin anddioxin-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 healthpolicy decisions (Gies et al., 2007; Steenland et al., 2001). Thus,chronic exposure to low/moderate doses of dioxin may beinvolved not only in the classic dioxin toxicity in somegenetically predisposed individuals (IARC Monograph, 1997;Aylward et al., 2005) but also in the newly identified role ofthese compounds in autoimmunity (Brembilla et al., 2011;Marshall and Kerkvliet, 2010; Ramirez et al., 2010).

In humans, skin lesions called ‘‘chloracne’’ are the mostvisible and consistent response to dioxin exposure and thereforeplay the role of a sentinel sign for toxicity (Caputo et al., 1988).The mechanism by which chloracne appears was not previouslyknown and its diagnostic value is not straightforward, especiallyin mild and sporadic cases, which could still relate to significantexposure (Passarini et al., 2010). A robust indicator that wouldtrigger specific ecotoxicology diagnostic processes is lacking; inthe current situation, it is likely that many cases have not beenrecognized (Saurat and Sorg, 2010).

We have previously reported on the TCDD poisoning inVictor Yushchenko with identification and measurement ofTCDD metabolites (Sorg et al., 2009). The maximum accepteddaily dose exposure in human is 4 pg/kg, and this patientreceived a single dose of 20 lg/kg.

With the approval of the patient to release peer-reviewedscientific information on his case, we now report on a set ofdata that has never been obtained in humans and helps definethe phenotype of the dioxin-induced skin pathology.

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: [email protected]

by guest on October 1, 2014

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… pour la Science

Histopathological and immunohistochemical evaluation. Sections werecut from formalin-fixed, paraffin-embedded skin biopsy specimens and stained

with hematoxylin and eosin. Cytochrome P450 (CYP) 1A1 immunohisto-

chemical analysis was performed with the use of an immunoperoxidase

technique according to standard procedures (Lebeau et al., 2005).

Dioxin analysis. TCDD was determined in lipid extracts from serum and

skin samples using gas chromatography with high-resolution mass spectrom-

etry as previously described (Sorg et al., 2009).

Imaging and volumetric analysis. Radiologic follow-up included high-

resolution magnetic resonance (MR) imaging examinations of the face and head

at 1.5 T and total body positron emission tomography/computed tomography

(PET/CT) exams. The acquired MR and PET/CT data obtained for diagnostic,pretherapeutic, and follow-up purposes formed the basis of our volumetric

analysis. The total skin volume and the combined volume of all hamartomatous

skin lesions in the examined area were calculated separately usingcommercially available software according to the manufacturers’ instructions

(GE Healthcare Advantage Windows). Individual skin lesions on each slice

were defined by manual contouring, and appropriate thresholding was used to

determine total skin volume. The software was used to generate three-dimensional representations of the calculated total skin volume and of the

calculated volume of all hamartomatous lesions.

Gene expression analysis. Total RNA extracts from skin samples (face) of

the patient and four healthy Caucasian men matched for age, sex, race, and site

of sampling were prepared as previously reported (Sorg et al., 2008). RNAquality was assessed using an Agilent 2100 Bioanalyzer with an RNA 6000

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 identifydifferentially expressed transcripts, comparisons were carried out after

normalization with the Affymetrix GCOS 1.2 (MAS5) software.

Bioinformatic analysis. Responsive elements for genes corresponding to

differentially expressed transcripts were searched with the University of

California—Santa Cruz Genome Browser (http://genome.ucsc.edu/, March2006 [NCBI36/hg18] assembly) in the Transcription Factor Binding Sites

Conserved Track (Fujita et al., 2010) and Gene2Promoter and MatInspector

from Genomatix software (www.genomatix.de) (Cartharius et al., 2005). Formost of the tested genes, the same responsive elements were found with bothresources.

RESULTS

Dioxin-Induced Skin Lesions Progress over Months whileInternal Organs Heal

A few hours after dinner in Kiev on 5 September 2004, a 50-year-old male patient suddenly became severely ill. Hospitalwork-up revealed gastritis, colitis with multiple ulcers, hepatitis,and pancreatitis, all compatible with poisoning by an ‘‘unknownsubstance’’ because testing for thallium, arsenic, antimony,mercury, and lead was negative (Ryan, 2011). Severe facialedema appeared 2 weeks after the poisoning (W2 a.p.). By W6a.p., the digestive tract symptoms had improved (Fig. 1).However, severe bilateral lower limb neuropathic pain appeared,and electroneuromyography confirmed that this was caused bysmall fiber peripheral neuropathy (The organs and systemsinvolved, other than the skin, are just cited here. Each will befully addressed in future publications when the mechanism hasbeen better analyzed by appropriate ongoing data analysis.).Facial involvement worsened with diffuse nodular lesions on anedematous background, sparing of the periocular zone, butmajor involvement of the ears and retroauricular folds (Figs. 2Aand 2B). The basic skin lesions were small nodules (Fig. 2C),

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.

LESSONS FROM THE POISONING OF VICTOR YUSHCHENKO 311



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included some genes specifically involved in sebum lipidmetabolism.

AhR-responsive elements were identified in the promoterregions of FADS2, AWAT1, ELOVL3, ALOX15B, and sterol O-acyltransferase 1 genes.

The differentially expressed genes involved in tissue renewingmay relate to hamartomas formation. Among the induced genes,gremlin 2 and high temperature requirement factor A serinepeptidase 3, which encode for antagonists of the bonemorphogenic protein family (BMP), may be highly relevant tothe process of hamartomas formation (Sneddon et al., 2006).

Most of the differentially expressed genes from structural/functional families shown in Table 1 as ‘‘extracellular matrix’’and ‘‘inflammation’’ were induced, which was expected fromthe clinicopathological but does not suggest a specific pattern.

The solute carrier family genes were all strongly repressedexcept one. This family encodes proteins involved in thetrafficking of many compounds including fatty acids, cholesterol,conjugated steroids, eicosanoids, peptides, and numerous drugs.That so many genes of this family/function were repressedsuggests that specific studies are indicated for analyzing the linkwith dioxin toxicity. This also applies to the differentiallyexpressed genes in the functional family of metabolism.

Dioxin Concentrates in the Skin Lesions: The Emergence ofa New Compartment

Figure 4 shows the progressive decrease of TCDD levels inthe serum, which contrasts with an increase of TCDD levels inthe skin lesions that reaches a peak at M11 a.p. At that time,TCDD concentration in skin lesions was 10-fold that of serum.This indicates that the skin lesions do constitute a significantnovel compartment, which we calculated to be for the face 482cm3 or 68.5% of the total skin volume (704 cm3) (Fig. 5).Because at M11 a.p. 40% of the total body skin surface wascovered with hamartomatous lesions, it can be estimated thatduring the peak manifestation of the cutaneous disease, thetotal volume of the hamartomatous compartment could havereached 6400 cm3 for the entire body, containing about 35 lgTCDD, i.e., 2% of the initial total body burden.

Synopsis of Therapeutical Measures Derived fromObservations

We initially observed that any incisional approach resultedin dystrophic healing, which we found to be a dioxin-induced,very fast skin healing response (Barouti et al., 2009). With thisimportant limiting feature to incisional skin intervention, wefound that mechanical dermabrasion and multiple micro punchextraction/aspiration techniques yielded very quick healing(again due to the mechanism cited above) and werecosmetically still satisfactory. These methods allowed bothsignificant pain relief from inflammation and extraction of largeamounts of dioxin-rich hamartomatous lesions. A total of 26procedures were performed on the whole-body skin surfaceunder general anesthesia, from M4 to M46 a.p.

Compassionate use of tumor necrosis factor a (TNF-a) blockadewas considered because non-steroidal anti-inflammatory drugs andsystemic steroids were not effective. The patient received threeinfusionsof infliximabbut becauseof intolerancewas then switchedto adalimumab, which was given for M18 (M16th to M34th a.p.).

FIG. 4. Kinetics of TCDD showing the concentration of dioxin in the skinlesions: the emergence of a new compartment. Despite a progressive decrease

in serum TCDD from months 3 to 15 a.p., there was a concomitant increase of

TCDD concentration in skin, while the surface of skin involvement spread. For

each parameter, the results are expressed as the percentage of the maximumvalues reached during this period of observation. The maximum values were as

follows: (A) for skin lesions: 40% of total body surface area at M11 a.p.

(evaluated prospectively by the palm of the hand method; Agarwal and Sahu,

2010); (B) for TCDD in serum: 890 pg/g wet weight (ww) at M3 a.p.; (C) forTCDD in skin lesions: 5000 pg/g ww at M11 a.p. At that time, TCDD

concentration in skin lesions was therefore 10-fold that of serum. This indicates

that the skin lesions do constitute a significant novel compartment, which couldhave reached 6400 cm3 for the entire body by M11 a.p., containing about 35 lg

TCDD, i.e., 2% of the total initial body burden (see Fig. 5 and discussion).

FIG. 5. Volumetric analyses of the skin lesions. (A) Three-dimensional

representation of the calculated total skin volume of the face using themethodology 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 lateralcheeks. (C) Thick slab reconstruction of an FDG PET data set obtained from

a whole-body PET/CT acquisition showing the distribution and the metabolism

of the skin lesions on the back of the patient. Posterior view. Note that some

lesions display a high metabolism (major FDG uptake, dark spots), whereasother lesions are metabolically less active (moderate FDG uptake, gray spots).

‘‘Metabolism’’ refers to FDG uptake, most probably linked to secondary

inflammation rather than to CYP1A1/dioxin metabolism.




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included some genes specifically involved in sebum lipidmetabolism.

AhR-responsive elements were identified in the promoterregions of FADS2, AWAT1, ELOVL3, ALOX15B, and sterol O-acyltransferase 1 genes.

The differentially expressed genes involved in tissue renewingmay relate to hamartomas formation. Among the induced genes,gremlin 2 and high temperature requirement factor A serinepeptidase 3, which encode for antagonists of the bonemorphogenic protein family (BMP), may be highly relevant tothe process of hamartomas formation (Sneddon et al., 2006).

Most of the differentially expressed genes from structural/functional families shown in Table 1 as ‘‘extracellular matrix’’and ‘‘inflammation’’ were induced, which was expected fromthe clinicopathological but does not suggest a specific pattern.

The solute carrier family genes were all strongly repressedexcept one. This family encodes proteins involved in thetrafficking of many compounds including fatty acids, cholesterol,conjugated steroids, eicosanoids, peptides, and numerous drugs.That so many genes of this family/function were repressedsuggests that specific studies are indicated for analyzing the linkwith dioxin toxicity. This also applies to the differentiallyexpressed genes in the functional family of metabolism.

Dioxin Concentrates in the Skin Lesions: The Emergence ofa New Compartment

Figure 4 shows the progressive decrease of TCDD levels inthe serum, which contrasts with an increase of TCDD levels inthe skin lesions that reaches a peak at M11 a.p. At that time,TCDD concentration in skin lesions was 10-fold that of serum.This indicates that the skin lesions do constitute a significantnovel compartment, which we calculated to be for the face 482cm3 or 68.5% of the total skin volume (704 cm3) (Fig. 5).Because at M11 a.p. 40% of the total body skin surface wascovered with hamartomatous lesions, it can be estimated thatduring the peak manifestation of the cutaneous disease, thetotal volume of the hamartomatous compartment could havereached 6400 cm3 for the entire body, containing about 35 lgTCDD, i.e., 2% of the initial total body burden.

Synopsis of Therapeutical Measures Derived fromObservations

We initially observed that any incisional approach resultedin dystrophic healing, which we found to be a dioxin-induced,very fast skin healing response (Barouti et al., 2009). With thisimportant limiting feature to incisional skin intervention, wefound that mechanical dermabrasion and multiple micro punchextraction/aspiration techniques yielded very quick healing(again due to the mechanism cited above) and werecosmetically still satisfactory. These methods allowed bothsignificant pain relief from inflammation and extraction of largeamounts of dioxin-rich hamartomatous lesions. A total of 26procedures were performed on the whole-body skin surfaceunder general anesthesia, from M4 to M46 a.p.

Compassionate use of tumor necrosis factor a (TNF-a) blockadewas considered because non-steroidal anti-inflammatory drugs andsystemic steroids were not effective. The patient received threeinfusionsof infliximabbut becauseof intolerancewas then switchedto adalimumab, which was given for M18 (M16th to M34th a.p.).

FIG. 4. Kinetics of TCDD showing the concentration of dioxin in the skinlesions: the emergence of a new compartment. Despite a progressive decrease

in serum TCDD from months 3 to 15 a.p., there was a concomitant increase of

TCDD concentration in skin, while the surface of skin involvement spread. For

each parameter, the results are expressed as the percentage of the maximumvalues reached during this period of observation. The maximum values were as

follows: (A) for skin lesions: 40% of total body surface area at M11 a.p.

(evaluated prospectively by the palm of the hand method; Agarwal and Sahu,

2010); (B) for TCDD in serum: 890 pg/g wet weight (ww) at M3 a.p.; (C) forTCDD in skin lesions: 5000 pg/g ww at M11 a.p. At that time, TCDD

concentration in skin lesions was therefore 10-fold that of serum. This indicates

that the skin lesions do constitute a significant novel compartment, which couldhave reached 6400 cm3 for the entire body by M11 a.p., containing about 35 lg

TCDD, i.e., 2% of the total initial body burden (see Fig. 5 and discussion).

FIG. 5. Volumetric analyses of the skin lesions. (A) Three-dimensional

representation of the calculated total skin volume of the face using themethodology 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 lateralcheeks. (C) Thick slab reconstruction of an FDG PET data set obtained from

a whole-body PET/CT acquisition showing the distribution and the metabolism

of the skin lesions on the back of the patient. Posterior view. Note that some

lesions display a high metabolism (major FDG uptake, dark spots), whereasother lesions are metabolically less active (moderate FDG uptake, gray spots).

‘‘Metabolism’’ refers to FDG uptake, most probably linked to secondary

inflammation rather than to CYP1A1/dioxin metabolism.




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22

with rapid growth into cysts that developed severe painfulinflammation. These lesions were clinically very similar to thoseof severe nodulocystic acne (Plewig and Kligman, 1993). ByW10 a.p., a few scattered cystic lesions had appeared on thebody, but it was not until 9 months (M9) a.p. that lesions becamewidespread over the entire body, including the limbs, witha peak clinical manifestation at M11 a.p (Fig. 1). Palms andsoles were spared and have remained so for the subsequent yearsof follow-up. Only a few lesions occurred in the axillary andinguino genital folds.

Skin Lesions Are Hamartomas

The data are based on 52 skin specimens taken over M43,which showed consistently identical aspects. The keyfeatures were ‘‘structure loss’’ and ‘‘structure gain,’’ withpreservation of other normal skin structures, therebycompatible with hamartomas (http://en.wikipedia.org/wiki/Hamartoma) (Albrecht, 1904).

The structure loss was the disappearance of the sebaceousglands. This loss occurred to a striking extent: on 252histological slides studied, not a single sebaceous gland wasobserved (a very conservative estimate of normal numberswould be from 500 to 4500 sebaceous glands observed on 252slides) (Montagna, 1963).

The structure gain was the appearance of cystic lesions withepithelial walls showing epidermal-like differentiation with theexpected distribution of epidermal keratins (not shown). Theselesions (Figs. 3A–C) were either superficial with an opencomedo-like aspect or extending much deeper in the dermis,hence resembling infundibular cysts (Plewig and Kligman,1993), but with the following distinct characteristics: (1)mantle-like columnar epithelial downgrowths, showing highproliferative activity (Ki67-positive cells, not shown), puta-tively giving birth to new cysts, resulting in branching cysticfigures with downward growth (Figs. 3A–C) and (2) focalexpression of CYP1A1, the major dioxin-metabolizing CYPenzyme, in the epithelial walls of the cystic lesions as shown byimmunohistochemistry (Figs. 3A and 3C).

Gene Expression in the Skin

Gene expression as monitored by whole-genome micro-arrays analysis was strongly altered in this hamartomatous skin,as compared with age, sex, race, and site of sampling skinspecimens taken in healthy subjects (Table 1).

The majority of differentially expressed genes were re-pressed: thus, when a cutoff of more than twofold wasconsidered for the analysis of skin samples taken at M5 a.p.,530 genes were found to be differentially expressed, 63% ofwhich were repressed.

Table 1 shows a more stringent sorting, with only the genesdownregulated more than 10-fold and those upregulated morethan threefold in skin samples taken M5 a.p.. Table 1 includes inaddition (1) the key genes of the aryl hydrocarbon receptor (AhR)dioxin signaling pathway, whatever their modulation, and (2)another transcriptome performed M11 a.p. in order to evaluatethe permanence of the altered gene expression, which was foundto be quite consistent (see Table 1, fold changes M5 and M11).

Among the genes known to be involved in AhR/dioxinpathway, the most induced were CYP1A1, CYP1B1, CYP1A2,and the aryl hydrocarbon receptor repressor (AhRR), whereasthe AhR itself, its chaperones heat shock protein 90 andprostaglandin E synthase 3 (p23), as well as aryl hydrocarbonreceptor nuclear translocator/HIFb genes were not differen-tially expressed. Real time PCR analysis for AhR, AhRR,CYP1A1, and CYP1A2, performed on independent samplesdissected at the same time, confirmed these results (data notshown). This indicates that a strong and sustained induction ofthe AhR pathway does occur in the skin lesions and provides,for the first time in humans, information on the relativeexpression of each player under high dioxin exposure.

The most represented structural/functional family of genesdifferentially expressed was related to lipid metabolism. In thisfamily, all genes were repressed except phospholipase A2group IIA, which was induced threefold. The repressed genes

FIG. 2. Representative pictures of the skin lesions. (A) Facial, auricular,

and retroauricular nodular lesions on an edematous background at month 4 a.p.

(B) Axial, high-resolution T2-weighted MR image at the level of the mid faceshowing many hamartomas located in the dermis, on the cheek, the ear lobe,

and retroauricular fold. Note variable size of the cysts, the largest being located

in the ear lobe. Some cysts extend into the subcutaneous fat. (C) Per-operative

aspect of the hamartomas on the ear lobule at month 4 a.p.

FIG. 3. Histological analyses of the skin lesions. (A) Photomicrograph ofa skin sample from the retroauricular area stained with hematoxylin-eosin (upper

part) showing the dermal distribution of the hamartomas and the absence of

sebaceous glands and (lower part) reacted with anti-CYP1A1 showing the focal

expression of the enzyme in the hamartomas (scanning magnification; monoclonalanti-CYP1A1 antibody, Santa Cruz Biotechnology, diluted 1:50). (B) Photomi-

crograph of one of the hamartoma showing mantle-like columnar epithelial

downgrowths (Hematoxylin-eosin, original magnification: X20). (C) Immunohis-

tochemical staining of CYP1A1 in a hamartoma. Original magnification: X20.

312 SAURAT ET AL.



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Prélèvements et analyses

PCB et composés dioxin-like

23

Ordonnance sur les travaux de construction

Obligation de planification des travaux si la présence de PCB est suspectée

24

fixe une limite de teneur de PCB dans les joints de

50 ppm (mg/kg)

Appliqué par extension aux autres matériaux (peintures)

25

Analyse des PCBs 27

PCB 52 PCB 180 PCB 138

PCB 28 PCB 101 PCB 153

Les PCB indicateurs les 6 grands électeurs

28

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$

%"m

asse"

1016$ 1242$ 1248$ 1248$ 1254$"Late"$ 1254$ 1260$

29

La composition des Aroclors

Données sources : ATDSR

28 52 101 138 153 180 Total % Facteur1016 8.50 4.63 0.04 13.2 7.61242 6.86 3.53 0.69 0.10 0.06 11.2 8.91248 3.59 6.93 2.22 0.38 0.23 0.02 13.4 7.51248 5.57 5.58 1.89 0.41 0.43 0.21 14.1 7.11254 "Late" 0.06 0.83 5.49 5.95 3.29 0.42 16.0 6.21254 0.19 5.38 8.02 5.80 3.77 0.67 23.8 4.21260 0.03 0.24 3.13 6.54 9.39 11.38 30.7 3.3

Aroclor

PCB

30 Les facteurs de conversion version massique

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

10161242 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-071248 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-071248 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-071254 "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-071254 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-071260 0.22 7E-06 0.48 1E-05 0.52 2E-05 0.02 6E-07 0.19 6E-06 0.1 3E-06

28 52 101 138 153 180 Total % Facteur1016 8.50 4.63 0.04 13.2 7.61242 6.86 3.53 0.69 0.10 0.06 11.2 8.91248 3.59 6.93 2.22 0.38 0.23 0.02 13.4 7.51248 5.57 5.58 1.89 0.41 0.43 0.21 14.1 7.11254 "Late" 0.06 0.83 5.49 5.95 3.29 0.42 16.0 6.21254 0.19 5.38 8.02 5.80 3.77 0.67 23.8 4.21260 0.03 0.24 3.13 6.54 9.39 11.38 30.7 3.3

Aroclor

PCB

Teq$ng/kg1016 01242 51248 391248 411254 "Late" 32441254 2171260 4

31 Et au niveau toxicité des PCB-dl ?

Teq (ng/g) pour 50 ppm

OK, les PCB indicateurs c’est

pourri,

mais comment on peut mesurer

un Teq TCDD ? La question que le premier rang se pose

Profil GC-HRMS d’un extrait de cendres volantes. © Restek

GC-HRMS 33

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 ?

Au secours DR CALUX !!!

Dr Heinrich von Calux 1875 - 1910

35 Peut-on mesurer réellement un

Teq TCDD alors ?

Dioxin Receptor Chemically Activated LUciferase gene eXpression

Méthode de biologie moléculaire de mesure de l’induction d’un récepteur par un ligand.

Vous allez voir, c’est tout simple

36

37

Voyez, c’est simple

38

NOYAU

Moins simple

39

… désolé

Principe du gène rapporteur (reporteur gene)

40

DR-CALUX méthodologie

41

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.

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

Mesure de la perméation des PCB indicateurs dans les combinaisons

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

12345678910

AverageMedian

Standard deviationCoeff. of variation

MinimumMaximum

RangeStnd. skewnessStnd. 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* 2928 2532 1820 1624 1620 1316* 1723 2119 3117 13

21.8 19.920 17.5

5.03 6.420.23 0.3216 1332 3116 18

1.34 0.980.31 -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

Exemples d’application du DR CALUX en environnement

Analyse des PCB dans des éléments en amiante-ciment

46

Concentration*TCDD-TEQ

Etat pg/g*TEQ pg/cm2*TEQ

Site*A*-*gris*clair <*0.067 <*0.12Site*A*-*gris*foncé*après*incendie 1.900 0.50Site*B*-*peinture*de*surface*rouge 0.940 2.70Site*C*-*gris*clair 0.400 0.34Site*D*-*gris*foncé 0.066 0.07Site*E*-*gris*foncé 0.037 0.05Site*F*-*gris*clair 0.029 0.03Site*G*-*gris*clair 0.061 0.05

Ech.%2 Ech.%3PCB$indicateurs pg/g pg/g

PCB'28 <5.8 <0.24PCB'52 <5.8 14

PCB'101 28 58PCB'138 103 57PCB'153 71 46PCB'180 141 13

Somme'des'7'PCB'indicateurs 344 188

Ech.%2 Ech.%3PCB$dioxin$like pg/g pg/g

PCB'77 <5.8 11PCB'105 81 29PCB'114 <5.8 <0.24PCB'118 49 30PCB'123 <5.8 <0.24PCB'126 <5.8 1.6PCB'156 <5.8 8.3PCB'157 <5.8 2.6PCB'167 <5.8 3.9PCB'169 <5.8 <0.24PCB'189 <5.8 <0.24

WHO(2005)'PCB'TEQ 2.5 0.19

1.72 ppm < 50 ppm

47

8 échantillons d’éléments en amiante-ciment (Suisse Romande)

TOXpro 2014, unpublished data

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)

A PECULIAR CASE OF PCB CONTAMINATION IN

YOUNG ORGANIC HENS

Wim Traag, Ron Hoogenboom, Guillaume van Dam, Jaap Immerzeel, Gerlof Oegema, Cornelis van der Kraats,

48

Exemples d’application du DR CALUX en hygiène du travail

Contamination des surfaces de travail aux composés Dioxin-like dans un incinérateur municipal

50 Prélèvement des surfaces (wipe-test)


Valeurs seuils Allemagne 10 ng/m2

0%#

10%#

20%#

30%#

40%#2378*TetraCDD#

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#1234678*HeptaCDF#1234789*HeptaCDF#

12346789*OctaCDF#

PCB#77#

PCB#81#

PCB#126#

PCB#169#

PCB#105#

PCB#114#

PCB#118#

PCB#123#

PCB#156#

PCB#157#

PCB#167#

PCB#189#

SIG$D13$($fines$sous$grille$

PCDD$

PCDF$

PCB'dl$

0%#

10%#

20%#

30%#

40%#2378*TetraCDD#

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#1234678*HeptaCDF#1234789*HeptaCDF#

12346789*OctaCDF#

PCB#77#

PCB#81#

PCB#126#

PCB#169#

PCB#105#

PCB#114#

PCB#118#

PCB#123#

PCB#156#

PCB#157#

PCB#167#

PCB#189#

SIG$D3$'$Cendres$volantes$

PCDD$

PCDF$

PCB'dl$

51

Recherche des sources par GC-HRMS

Cendres volantes Dépôt sur armoire


Mais bon, y’a pas que les PCB / Dioxines

dans la vie ! PCB et composés dioxin-like

Les retardateurs de flamme

Les retardateurs de flamme

53

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

Effect profiling of compounds with CALUX panel

Assay

Com

pound

Each compound has a different in vitro effect profile ? Is it possible to relate these profiles to in vivo toxicity of the compounds?

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

compound ‘profile’

CALUX assays currently available:

55 La gamme des récepteurs et « pathway)

56 Les possibilités de screening

Screening des micropolluants, eaux uséés

57

OUF !

58

dioxines, pcb, furanes et dr-calux

Health & Medicine