title: Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down.
abstract: BACKGROUND
  2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood.
  
  
  OBJECTIVE
  To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers.
  
  
  METHODS
  Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E.
  
  
  RESULTS
  In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype.
  
  
  CONCLUSIONS
  Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype.
authors.affiliation: Dermatological Sciences, 2nd Floor Leech Building, Medical Science, Institute of Cellular Medicine, Newcastle University, NE2 4HH, United Kingdom. Electronic address: a.forrester@tigem.it.
authors.forename: Alison R
authors.initials: AR
authors.lastname: Forrester
authors.affiliation: Dermatological Sciences, 2nd Floor Leech Building, Medical Science, Institute of Cellular Medicine, Newcastle University, NE2 4HH, United Kingdom. Electronic address: m.s.elias@newcastle.ac.uk.
authors.forename: Martina S
authors.initials: MS
authors.lastname: Elias
authors.affiliation: Dermatological Sciences, 2nd Floor Leech Building, Medical Science, Institute of Cellular Medicine, Newcastle University, NE2 4HH, United Kingdom. Electronic address: e.l.woodward@ncl.ac.uk.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Woodward
authors.affiliation: Global Safety Assessment, Alderley Park, AstraZeneca, Cheshire, United Kingdom. Electronic address: mark.j.graham@me.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Graham
authors.affiliation: Toxicology, Institute of Cellular Medicine, Newcastle University, NE1 7RU, United Kingdom. Electronic address: faith.williams@ncl.ac.uk.
authors.forename: Faith M
authors.initials: FM
authors.lastname: Williams
authors.affiliation: Dermatological Sciences, 2nd Floor Leech Building, Medical Science, Institute of Cellular Medicine, Newcastle University, NE2 4HH, United Kingdom. Electronic address: nick.reynolds@ncl.ac.uk.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Reynolds
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D054506:Chloracne / Q000209:etiology* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D004817:Epidermis / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000091344:Filaggrin Proteins; D055785:Gene Knockdown Techniques; D006801:Humans; D007211:Indoles / Q000633:toxicity; D007381:Intermediate Filament Proteins / Q000378:metabolism; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008024:Ligands; D010641:Phenotype; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D011498:Protein Precursors / Q000378:metabolism; D034622:RNA Interference; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000633:toxicity; D014162:Transfection; D011503:Transglutaminases / Q000378:metabolism; D019324:beta-Naphthoflavone / Q000633:toxicity
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D019363:Cytochrome P-450 CYP1A1; D000091344:Filaggrin Proteins; D007211:Indoles; D007381:Intermediate Filament Proteins; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D011498:Protein Precursors; D018336:Receptors, Aryl Hydrocarbon; D013844:Thiazoles; D011503:Transglutaminases; D019324:beta-Naphthoflavone; C548651:2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester; C111235:AHR protein, human; C000717249:FLG protein, human; C032253:involucrin; C569463:CYP1A1 protein, human; C105804:transglutaminase 1
keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin; 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester; AHRR; AhR; Aryl hydrocarbon receptor; CYP1A1; Epidermal equivalent; ITE; Keratinocyte; TCDD; TGM-1; XRE; aryl hydrocarbon receptor; aryl hydrocarbon repressor protein; cytochrome P450 1A1; transglutaminase-1; xenobiotic response element; α-NF; α-naphthoflavone; β-NF; β-Naphthoflavone; β-naphthoflavone