Elsevier

Toxicology in Vitro

Volume 24, Issue 3, April 2010, Pages 973-980
Toxicology in Vitro

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

https://doi.org/10.1016/j.tiv.2009.12.023Get rights and content

Abstract

Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n = 4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 μM) induced EROD activities of 19.0 ± 0.9 pmol/mg/min (n = 11) in HaCaT and 5.8 ± 0.5 pmol/mg/min (n = 4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8 ± 0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0–44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.

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)

  • B. Hirel et al.

    In vitro expression of drug metabolizing enzyme activities in human adult keratinocytes under various culture conditions and their response to inducers

    Toxicol. In Vitro

    (1995)
  • A. Janmohamed et al.

    Quantification and cellular localization of expression in human skin of genes encoding flavin-containing monooxygenases and cytochromes P450

    Biochem. Pharmacol.

    (2001)
  • B. Lehmann et al.

    Human keratinocyte line HaCaT metabolizes 1alpha-hydroxyvitamin D3 and vitamin D3 to 1alpha, 25-dihydroxyvitamin D3 (calcitriol)

    J. Dermatol. Sci.

    (1998)
  • A. Meyer et al.

    Determination of p-phenylenediamine and its metabolites MAPPD and DAPPD in biological samples using HPLC-DAD and amperometric detection

    J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

    (2009)
  • R.F. Minchin et al.

    Arylamine N-acetyltransferase I

    Int. J. Biochem. Cell Biol.

    (2007)
  • R. Moeller et al.

    Impact of para-phenylenediamine on cyclooxygenases expression and prostaglandin formation in human immortalized keratinocytes (HaCaT)

    Toxicology

    (2008)
  • D.W. Nebert et al.

    Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer

    J. Biol. Chem.

    (2004)
  • T.P. Reilly et al.

    A role for bioactivation and covalent binding within epidermal keratinocytes in sulfonamide-induced cutaneous drug reactions

    J. Invest. Dermatol.

    (2000)
  • E.W. Scholes et al.

    Skin metabolism of contact allergens

    Toxicol. In Vitro

    (1994)
  • S. Sieben et al.

    Characterization of T cell responses to fragrances

    Toxicol. Appl. Pharmacol.

    (2001)
  • J.C. Sinclair et al.

    Purification, characterization, and crystallization of an N-hydroxyarylamine O-acetyltransferase from Salmonella typhimurium

    Protein Expr. Purif.

    (1998)
  • F.M. Van Och et al.

    Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1alpha and IL-18 production by a murine and human keratinocyte cell line

    Toxicology

    (2005)
  • R.J. Vandebriel et al.

    In vitro assessment of sensitizing activity of low molecular weight compounds

    Toxicol. Appl. Pharmacol.

    (2005)
  • P.H. Villard et al.

    Increase of CYP1B1 transcription in human keratinocytes and HaCaT cells after UV-B exposure

    Toxicol. Appl. Pharmacol.

    (2002)
  • T. Al Masaoudi et al.

    Eugenol but not isoeugenol induces CYPIA1 mRNA in human keratinocytes

    Int. Arch. Allergy Immunol.

    (2001)
  • D.F. Barker et al.

    Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1

    Pharmacogenet. Genomics

    (2006)
  • B. Blomeke et al.

    Elicitation response characteristics to mono- and to N,N′-diacetyl-para-phenylenediamine

    Contact Dermatitis

    (2008)
  • S. Boukouvala et al.

    Structural analysis of the genes for human arylamine N-acetyltransferases and characterisation of alternative transcripts

    Basic Clin. Pharmacol. Toxicol.

    (2005)
  • Cited by (0)

    View full text