All statistical analysis was performed using Graphpad Prism 5. Different methods were used between luciferase assays and Western blots because Westen blots showed that time became an important factor. Statistical analysis was not performed on preliminary data because sample size (number of donors that each assay was replicated in) was too low.
Luciferase assays
Mean values were taken from triplicate wells for each donor. Values were normalised by Log transformation for analysis. One-way ANOVAs were performed for each ligand, comparing vehicle to each dose, then for inhibition assays, ligand at each dose was compared to both concentrations of α-NF by one-way ANOVA. This answered two questions: the effect of ligand and the effect of α-NF on ligand responses. Post hoc tests used were Dunnetts or analysis of linear trend chosen depending on the linearity of the dose response. Dunnetts post test was used to compare vehicle to each separate dose when no linear dose response was observed. Analysis of liner trend was
performed to measure dose responses when present.
Western blots
Densitometry was carried out on representative blots from 3 donors and normalised to β-actin. Values were normalised by Log transformation for analysis. Data sets showing the effects of AhR agonist on protein levels were analysed using two-way ANOVA, comparing ligand dose and time. Inhibition studies were also analysed by two-way ANOVA, comparing the effects of α-NF and time. Post hoc tests were not performed for two-way ANOVAs.
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3 AhR activation in primary human keratinocytes
3.1 Introduction
3.1.1 Known effects of AhR activation
The AhR is the mediator of a wide range of toxicities from developmental defects to neurological disorders and cancers (Gasiewicz et al., 2008; Singh et al., 2009a; Williamson et al., 2005). AhR activation by TCDD mediates the well known human toxicity chloracne, an acne-like skin lesion resulting from exposure to TCDD
(Panteleyev and Bickers, 2006; Sorg, 2009). Chloracne is defined and described in detail in the introduction (section 1.3). The defining characteristics of a chloracnegen are unknown but some hypotheses are that high-residency of the ligand or high-affinity for the AhR are required to cause chloracne. To study this further, we used three ligands with different characteristics: chloracnegenic TCDD which displays high-affinity and residency (Table 4)(Schwetz et al., 1973; Sorg, 2009), low-affinity exogenous agonist β-NF that is not chloracnegenic (see section 1.1.4.2)(Maier et al., 1998; Song et al., 2002) and highly potent physiological agonist ITE that is not known to be
chloracnegenic but has not been fully characterised in skin or models of chloracne (section 1.1.4.2)(Henry et al., 2006; Henry et al., 2010; Song et al., 2002). The characteristics of these agonists are described in detail in the introduction (section 1.1.4.2).
AhR affinity Residency Metabolism Potency
TCDD (nM) +++ +++ + +++
β-NF (µM) + + +++ +
ITE (µM) ++ + +++ ++
Table 4. The characteristics of AhR-agonists TCDD, β-NF and ITE.
Plus sign represents strength of characteristic for each ligand. Information is taken from the literature as described in Chapter 1.
AhR is known to induce wide ranging effects including immunosupression (Lundberg et al., 1991; Matulka et al., 1997) and anti-inflammatory effects (Benson and Shepherd, 2011; Wu et al., 2011). There are tissue specific effects intraspecies variation in AhR responses that include variation of AhR degradation (eg. low AhR activation in mouse fibroblasts and high AhR activation in embryonic carcinoma)(FitzGerald et al., 1996)
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and induced migration in MCF-7 cells, caused by autocrine activity of CTSD (Ohri et al., 2008; Seifert et al., 2009). Levels of AhR response are dependent in part on expression of the AhR (Dolwick et al., 1993; Mason and Okey, 1982) and also the AhR repressor protein (AhRR) (which is induced by AhR activation) as described in the introduction (section 1.1.2). For example, human fibroblasts are thought to show low AhR activity because of low expression of AhR and high expression of AhRR (Akintobi et al., 2007; Gradin et al., 1999). The effects of AhR activation also appear to be species specific; the differences are thought mainly to be caused by variation in affinity of the AhR to the ligand (Henry et al., 1989; Ramadoss and Perdew, 2004; Schwetz et al., 1973). For example, Perdew and Glover showed forms of AhR with different ligand affinity in mouse strains (Poland and Glover, 1990), while Ramadoss and Perdew showed that AhR affinity in humans was lower than mouse AhR in competition binding experiments (Ramadoss and Perdew, 2004).
As discussed in the introduction (section 1.1.1) CYP1A1 is a commonly used readout of AhR activation and potential for chloracnegenic toxicity. During drug development in industry all novel compounds are screened for causing CYP1A1 induction. However, CYP1A1 expression may not correlate directly to AhR induced toxicity. Thus
compounds such as β-NF may induce CYP1A1 in specific cell types yet β-NF is known not to have chloracnegenic potential. One aim of the studies described was to test whether that the different characteristics of these ligands would correlate with different downstream effects in cultures keratinocytes and cutaneous models, suggesting toxicity specific markers.
The first step in the project was to characterise AhR activation in primary keratinocyte monolayers, justifying dose ranges and time points and forming treatment regimes to put into the epidermal equivalent model. This model is important in studying the AhR because a) TCDD induces changes in keratinocyte differentiation that appear relevant to chloracne in this model and b) the effects of confluency on AhR localisation (Ikuta et al., 2004; Sadek and Allen-Hoffmann, 1994a, b) and the effects of differentiation (Du et al., 2006a) on AhR activation in keratinocyte monolayer culture. Studies in monolayer culture therefore required carefully controlled and defined conditions.
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3.1.1.1 Comparisons of AhR agonists in the literature
Comparisons of AhR activation by TCDD, ITE and β-NF have been previously reported in the literature. Du et al utilised keratinocyte cultures differentiated in high calcium medium to compare the AhR mediated effects of β-NF and TCDD treatment on proliferating or differentiating keratinocytes over 6 days (Du et al., 2006a). CYP1A1 protein was induced to similar levels at 6 to 8 days by 30µM β-NF and 10nM TCDD, with AhR degradation observed in parallel. TCDD induced more AhR degradation than β-NF. Du et al showed up-regulation of TGM-1 expression and TGM activity by TCDD and not β-NF, concluding that this could be a contributing factor of TCDD induced chloracne (Du et al., 2006a). Keratinocytes were cultured in high calcium medium for 16 days and were shown to developed a basic type of 3D culture (Du et al., 2006b).
However this model did not include media supplements of vitamin C or an air liquid interface to allow cornification of keratinocytes (as in epidermal equivalents used in this project or by Loertscher or Geaseau, details shown in introduction and next chapter). Data complementary to these papers is presented in this thesis; I have shown studies in monolayer keratinocyte cultures treated with TCDD and β-NF, including novel data on ITE in keratinocytes and comparisons between TCDD/β-NF and ITE. I also present novel data on the treatment of epidermal equivalents with ITE (and β-NF which has not been studied in epidermal equivalents in regards to its phenotypic effects) and again compare the effects to TCDD.
ITE has been recently studied and compared to TCDD and β-NF to define its properties but no previous studies have investigated ITE in keratinocytes or epidermal equivalents. Song et al compared levels of AhR activation induced by 100nM – 10µM ITE or 10nM – 100µM β-NF in a mouse hepatoma cell line and also used isolated murine and human AhR for in vitro binding studies (Song et al., 2002). Binding studies showed that 0.5 and 5µM ITE could compete against 2nM TCDD for AhR binding. The authors concluded that binding affinities of β-NF and ITE for the AhR were similar whereas binding affinity of TCDD for the AhR was slightly higher (ki (nM) for murine AhR: ITE = 3, β-NF = 2, TCDD = 0.5 ). Based on luciferase assays in a mouse hepatoma cell line, potencies were different; µM ITE was 5 times more potent than the same concentrations of β-NF. They hypothesise that this may have been due to greater stability of ITE (compared to β-NF), that ITE may have greater access to the nucleus or that it may induce
conformational changes that causes AhR to be more efficient at binding DNA. Henry et al. studied levels of AhR activity induced in Hepa cells by a range of concentrations of
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ITE and TCDD (1-5000nM ITE and 0.01 – 1nM TCDD) by luciferase assay and Western blot (Henry et al., 2006). The XRE-luciferase activity was induced to similar levels by both ligands (at doses of ITE 1000 fold higher than TCDD) and AhR activity was measured by Western blot (down-regulation of AhR and up-regulation of CYP1A1), showing similar levels of activation by 0.1 – 1 nM TCDD and 0.1 – 5µM ITE. Differences were seen in that TCDD induced sustained activation, where as ITE-treated samples showed partial AhR recovery by 24h. Conclusions drawn from this paper are that potency of ITE (at doses 1000 fold higher than TCDD) was equal to that of TCDD. Rapid AhR protein recovery over 24 h was thought to be caused by low stability of ITE. Very short time points were used in the study by Henry et al (Henry et al., 2006)(4 h and 24 h), so comments on sustained activation by TCDD compared to ITE were only over 24h. It would have been interesting if this time course had been expanded further to show effects over prolonged treatment. Further studies were carried out by Henry et al. (Henry et al., 2010) in primary mouse lung fibroblasts and produced similar results as in (Henry et al., 2006), showing equal activation induced by 0.25 – 2.5µM ITE and 0.2 – 1nM TCDD (Henry et al., 2010). The Henry and the Song papers report data on ITE compared to TCDD (Henry et al., 2006; Henry et al., 2010) and ITE compared to β-NF (Song et al., 2002) in mouse hepatoma cell lines. Comments are made on the
comparable potencies of the agonists; TCDD and ITE have similar potencies but prolonged activation is only induced by TCDD due to the relatively fast metabolism of ITE (Henry et al., 2006); the Song paper compares ITE to β-NF and reports that ITE has a similar affinity for the AhR as β-NF (reporting murine ki: ITE = 3, β-NF = 2, TCDD = 0.5nM) but that ITE has a higher potency due to its increased stability (compared to β- NF). Neither paper references any metabolic studies of ITE. It is presumed that as a physiological agonist, it must be relatively quickly cleared to act efficiently as part of a signalling pathway.
These doses were chosen based on the literature in keratinocytes, a relevant treatment model in a rat hepatoma cell line (Du et al., 2006a; Khan et al., 1992; Song et al., 2002) and epidermal equivalents (Geusau et al., 2005; Loertscher et al., 2001a; Loertscher et al., 2001b). ITE had not been tested in keratinocytes or epidermis, so the
concentrations utilised in experiments were based on the comparisons by (Henry et al., 2006; Song et al., 2002). Different time courses were used in hepatic cell lines and epidermal cultures to show optimal AhR activation in literature; In hepatic cell lines Western blots were performed at 24h and luciferase assays at 1h -24h showing a peak
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between 4 and 8 h (Henry et al., 2006; Song et al., 2002), whereas in differentiating keratinocyte cultures, Western blots were performed at time points up to 8 days. Du et al show low induction of CYP1A1 at day 6 (TCDD and β-NF treatment) and stronger induction at day 8, so this suggests that protein effects of AhR activation in the skin are not seen until then (Du et al., 2006a). To test this theory I included time points at days 2 and 4 in preliminary luciferase studies (Figure 3.2 A). Henry et al. show luciferase assay peaks between 4 and 8 h, so to test if there was early activation in keratinocytes, we included an 8 h time point in our luciferase studies (Figure 3.2). We show novel comparisons of AhR ligands TCDD, β-NF and ITE in their ability to activate AhR transcriptional activation and effects on AhR and CYP1A1 protein levels in primary keratinocytes.