Abstract
Background2,3,7,8-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. ObjectiveTo elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. MethodsUsing 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. ResultsIn 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. ConclusionLigand-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.
Highlights
The aryl hydrocarbon receptor (AhR) is a highly conserved member of the basic helix-loop-helix PER/ARNT/SIM family of transcription factors. It is activated by a wide range of planar polycyclic aromatic hydrocarbons (PAH) or halogenated aromatic hydrocarbons (HAH), including the highly potent ligand 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)
By comparing the effects of chloracnegenic and non-chloracnegenic AhR ligands on AhR activation and on the phenotype and differentiation of keratinocytes in an epidermal equivalent model, we show that the chloracne-like phenotype depends on AhR activation whereas AhR knock down is not sufficient to induce the phenotype
Agonist TCDD and the non-chloracnegenic AhR agonists b-NF and ITE on AhR activation in normal human epidermal keratinocytes (NHEK) and to determine dosimetry for use in epidermal equivalents, transcriptional activation by XREluciferase assay, cytochrome P450 (CYP1A1) induction and AhR degradation were initially studied in primary NHEK monolayer cultures
Summary
The aryl hydrocarbon receptor (AhR) is a highly conserved member of the basic helix-loop-helix PER/ARNT/SIM family of transcription factors. Because of significant differences between rodent and human skin and the lack of suitable animal models, 3-D human skin equivalent models have been evaluated for the further study of chloracne pathogenesis These models are not currently advanced enough to contain structures such as sebaceous glands, nor do they contain the potential to develop cysts, TCDD-treated epidermal equivalents have been shown to develop key features found in chloracne histology including: (1) hyperkeratinisation of the stratum corneum and (2) a thinner viable cell layer [15]. During drug development, screening for chloracnegenic potential of novel compounds is performed but current assays are based on CYP1A1 induction in non-keratinocyte cell lines
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