Evaluation of cytochrome P450 1 (CYP1) and N-acetyltransferase 1 (NAT1) activities in HaCaT cells: Implications for the development of in vitro techniques for predictive testing of contact sensitizers
Introduction
For animal welfare reasons as well as to meet the regulatory demands in Europe, there is a growing pressure to reduce the reliance on animal experiments for hazard identification. Regarding the toxicological end point of skin sensitization in particular, there is an insistent requirement to develop non-animal and in vitro alternatives, since the European Union will ban in vivo testing of cosmetic and toiletry ingredients in 2013 according to the 7th Amendment to the Cosmetic Directive. However, at present, validated strategies (reviewed in (Gerberick et al., 2007)) to identify sensitizing chemicals and evaluate their potency still rely on in vivo methods (Kimber et al., 2001).
To model the complex biology of immune responses with non-animal or in vitro skin sensitization assays, epidermal biotransformation may be considered for certain chemicals, since some of them are not protein reactive per se and may need metabolic conversion. Biotransformation generally plays a role in elimination of compounds but in certain instances metabolism may lead to the generation of intermediates capable of protein haptenation (Lepoittevin, 2006, Gerberick et al., 2009) and sensitization. Relevant xenobiotic metabolizing enzymes are expressed by both antigen-presenting cells (Sieben et al., 1999, Sanderson et al., 2007, Lichter et al., 2008) and keratinocytes (Hirel et al., 1995, Kawakubo et al., 2000, Baron et al., 2001, Saeki et al., 2002). The latter have been shown to be involved in bioactivation (Reilly et al., 2000) and conversely in deactivation of chemicals including drugs (Vyas et al., 2006a) and contact allergens (Kawakubo et al., 2000).
Several cell-based approaches to assess key steps in initial skin sensitization are under development and evaluation. The majority of them are using solely antigen-presenting cells such as blood-derived dendritic cells or human myeloid cell lines (Dos Santos et al., 2009). Lately, Schreiner and co-workers proposed a loose-fit coculture assay using dendritic cell-related monocytes and primary keratinocytes (Schreiner et al., 2007), which may contribute to immune responses besides danger signaling (Vandebriel et al., 2005) also by metabolic conversion of the added chemical. In order to avoid limitations caused by cell isolation, cost intensive primary cell culture as well as donor dependencies, the replacement of human primary keratinocytes by immortalized keratinocytes such as the cell line HaCaT may be a promising tool.
Knowledge about xenobiotic metabolizing enzyme activities in HaCaT cells is very limited (Lehmann et al., 1998, Goebel et al., 2009) but it has been demonstrated that HaCaT cells are able to activate certain drugs such as sulfamethoxazole (SMX) and dapsone (DDS) (Roychowdhury et al., 2005), whose hydroxylated derivatives adducted cellular proteins (Reilly et al., 2000). Although some studies addressed comparative analysis of cytochrome P450 mRNA in primary and HaCaT keratinocytes (Janmohamed et al., 2001, Villard et al., 2002, Vyas et al., 2006a) CYP1 enzyme activities are rarely studied in those cells (Cotovio et al., 1997, Delescluse et al., 1997, Harris et al., 2002). Regarding phase 2 metabolism, N-acetylation is known to deactivate certain skin allergens such as para-phenylenediamine (PPD) (Sieben et al., 2001b, Blomeke et al., 2008, Aeby et al., 2009) and previous data demonstrated that HaCaT keratinocytes are able to N-acetylate arylamine compounds (Moeller et al., 2008, Goebel et al., 2009).
Early in vivo data already suggested the involvement of xenobiotic metabolizing enzymes belonging to the CYP1 family in activation of chemical contact allergens (Scholes et al., 1994). Recent in vitro metabolism studies emphasize that hydroxylating enzyme activities (Vyas et al., 2006b) including CYP1 (Ott et al., 2009) as well as N-acetylation by NAT1 (Aeby et al., 2009) can play an important role in the generation of immunoreactive intermediates and detoxification of immunogens, respectively.
However, a detailed analysis of the cell line HaCaT and primary keratinocytes regarding CYP1 activities and N-acetylation in parallel has not yet been performed. We therefore analyzed CYP1 activities, N-acetylation capacities and promoter dependent NAT1 mRNA expression of HaCaT cells obtained from three different shipments (referred to as HaCaT A, B and C) and neonatal normal human epidermal keratinocytes (NHEK) from 5 individual donors.
Section snippets
Chemicals and reagents
para-phenylenediamine (1,4-diaminobenzene, PPD, purity ⩾ 99%), para-aminobenzoic acid (PABA, purity ⩾ 99%), 2-aminofluorene (AF, purity ⩾ 98%), dithiothreitol (DTT, purity ⩾ 99%), 4-dimethylaminobenzaldehyde (DMAB, purity ⩾ 99%), benzo[a]pyrene (B[a]P, purity > 97%), acetonitrile (high-performance liquid chromatography [HPLC] grade), resorufin (purity 95%), ethoxyresorufin (purity 99.5%), dicumarol (purity > 99%) and Bradford reagent were purchased from Sigma–Aldrich (Taufkirchen, Germany). Acetyl-Coenzyme A
Ethoxyresorufin O-deethylase (EROD) activity of HaCaT and primary keratinocytes
In order to analyze HaCaT keratinocytes from three different shipments (referred to as HaCaT A, B and C) and neonatal normal human epidermal keratinocytes (primary keratinocytes, NHEK) for EROD (CYP1) activities, we treated the cells with 1 μM B[a]P for 24 h. Initial experiments and existing literature (Harris et al., 2002) showed, that inducibility of EROD activities increased with confluence in HaCaT and primary keratinocytes and therefore, EROD activities were measured after the cells reached
Discussion
Prediction of skin metabolism, sensitization hazard and potency of chemicals are essential aspects in the development of non-animal assays as alternative in vitro testing methods. Such methods need to comprise the different stages of the multistep process of skin sensitization. Oxidative skin metabolism and N-acetyltransferase mediated acetylation are involved in the elimination of compounds but in some instances metabolism may lead to bioactivation and generation of intermediates capable to
Conflict of interest statement
None declared.
Acknowledgements
This work has been partly financed by “Landesgraduiertenförderung Rheinland-Pfalz”, Germany and by Wella Service GmbH, Darmstadt, Germany.
References (56)
- et al.
Skin Sensitization to p-penylenediamine: the diverging roles of oxidation and N-acetylation for dendritic cell activation and the immune response
J. Invest. Dermatol.
(2009) - et al.
Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes
J. Invest. Dermatol.
(2001) - et al.
Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types
Toxicol. In Vitro
(1997) - et al.
Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound
Toxicol. Appl. Pharmacol.
(2009) - et al.
Determination of 4-amino-m-cresol and 5-amino-o-cresol and metabolites in human keratinocytes (HaCaT) by high-performance liquid chromatography with DAD and MS detection
J. Biochem. Biophys. Methods
(2004) - et al.
Skin metabolism of aminophenols: human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo
Toxicol. Appl. Pharmacol.
(2009) - et al.
Induction of CYP1A1 by serum independent of AhR pathway
Biochem. Biophys. Res. Commun.
(2000) - et al.
Serum induces a transcriptional activation of CYP1A1 gene in HepG2 independently of the AhR pathway
Life Sci.
(2001) - et al.
Levels of cytochrome P-450-mediated aryl hydrocarbon hydroxylase (AHH) are higher in differentiated than in germinative cutaneous keratinocytes
J. Invest. Dermatol.
(1990) - et al.
Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens
Toxicol. Appl. Pharmacol.
(2008)