2,3,7,8-tetrachlorodibenzo-p-dioxin impairs differentiation of normal human epidermal keratinocytes in a skin equivalent model.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that is taken up via the food chain and accumulates in fat tissue. In humans, intoxication with TCDD causes chloracne, a severe skin disease resulting in disfiguring scarring (Geusau et al, 1999, 2001). In the past, various experimental approaches have been adapted to study the effect of TCDD on keratinocytes (KC) and skin. In utero exposure of murine fetuses led to an accelerated terminal differentiation in fetal mouse skin (Loertscher et al, 2002). Treatment of non-transformed human KC cultures with TCDD resulted in enhanced differentiation, determined by an increase of involucrin expression and modulation of expression of a number of growth-regulatory proteins (Gaido and Maness, 1994). One study on the effects of TCDD was performed with a spontaneously immortalized human KC cell line in a three-dimensional (3D) skin equivalent (SE) model, where TCDD caused acceleration of differentiation as determined by morphological characterization and by immunohistochemistry for several differentiation-specific protein markers; neither an influence on proliferation nor an induction of apoptosis was observed (Loertscher et al, 2001). In this study, we investigated the effect of TCDD on normal human epidermal KC grown in a 3D model under differentiating conditions (Stark et al, 1999; Rendl et al, 2002). After exposure of SE to TCDD at concentrations of 10 8 and 10 7 M for 1 wk, we quantified alterations of the KC differentiation process by western blot analysis with antibodies against selected differentiation markers. In vitro reconstructed SE were generated according to the protocol of Stark and colleagues with modifications described previously (Stark et al, 1999; Rendl et al, 2002). Starting on the day of lifting the culture to the air interface, culture medium for SE was supplemented with either TCDD (AccuStandard Inc., New Haven, Connecticut) (10 7 and 10 8 M) or vehicle every 2 d and culture was continued for up to 7 d. After 3 and 7 d of culture samples were taken by punch biopsy for hematoxylin & eosin staining and immunohistochemistry, performed as described previously. SE were also lyzed in a buffer containing 6 M urea 62.5 mM Tris/HCl, pH 6.8, 2% SDS, and 10% glycerol and subjected to western blot analysis as described previously (Rendl et al, 2002). The following antibodies were used at the indicated concentrations: goat polyclonal anti-CYP1A1 (Gentest Corporation, Woburn, Massachusetts) 1:500, mouse monoclonal anti-involucrin (NeoMarkers, Fremont, California) 1:1000, mouse monoclonal anti-filaggrin (Biomedical Technologies, Stoughton, Massachusetts) 1:500, rabbit polyclonal anti-Id-1 (Santa Cruz Biotechnology, Heidelberg, Germany) 1:1000, mouse monoclonal anti-p63 (Oncogene Research Products, Boston, Massachusetts) 1:300, mouse monoclonal anti-p16 (NeoMarkers) 1:250, mouse monoclonal anti-K1/10/11 (Cymbus Biotechnology, Hampshire, UK) 1:1000, and a rabbit polyclonal antibody that we had raised against recombinant human caspase-14 (Alibardi et al, in press). The most prominent effect of TCDD was an increase of thickness of the stratum corneum with parakeratosis and a reduction of viable cell layers after 7 d of treatment, compared with vehicle-treated samples (Fig 1). A complete loss of the granular layer was visible both on days 3 and 7 of incubation with TCDD. For quantitative comparison of SE treated and untreated with TCDD, the abundance of selected marker proteins was determined by western blotting. Expression of cytochrome P450 1A1 (CYP1A1), a dioxin-responsive gene (Xu et al, 2000), was strongly induced in a concentration-dependent manner (Fig 2), indicating the validity of the model used. TCDD decreased expression of p63 and Id-1, transcription factors that are downregulated during terminal KC differentiation (Langlands et al, 2000; Koster and Roop, 2004), at a concentration of 10 8 M and virtually abrogated their expression at 10 7 M. In line with previous reports on overexpression of Id-1 in KC (Nickoloff et al, 2000), modulation of the amount of Id-1 was accompanied by an inverse regulation of p16 that was concentration-dependently upregulated by TCDD (Fig 2). Expression of markers of KC differentiation was heterogenous: whereas the abundance of involucrin was increased by TCDD, the levels of keratins 1 and 10 were decreased after 7 d of TCDD treatment (Fig 2). Caspase-14, a protease implicated in terminal differentiation (Eckhart et al, 2000), was downregulated by TCDD in a concentration-dependent manner. Additionally, processing of pro-caspase-14 to the mature enzyme subunits was readily detectable in control SE but was not observed after TCDD treatment. Western blot analysis of profilaggrin/filaggrin revealed an increase of the abundance of profilaggrin under TCDD exposure. But processing of profilaggrin into mature filaggrin, one of the final steps of KC differentiation, was completely blocked by TCDD. Abbreviations: KC, keratinocytes; SE, skin equivalent; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin