Characterization of enzyme activities of Cytochrome P450 enzymes, Flavin-dependent monooxygenases, N-acetyltransferases and UDP-glucuronyltransferases in human reconstructed epidermis and full-thickness skin models
Introduction
The skin is exposed to a series of xenobiotics such as cosmetics and their ingredients. The 7th Amendment to the European cosmetic directive of March 2009 prohibits the use of animals for cosmetic testing hence alternative methods are urgently demanded to assess their toxic potential. One approach is the use of commercially available reconstructed skin models. These 3-dimensional organotypic tissues, made of human keratinocytes and fibroblasts, are being investigated for several years in context of testing corrosive or irritating potential of substances (Netzaff et al., 2005, Liebsch et al., 2009, OECD, 2004a, OECD, 2004b). While enzymatic capacities of skin (reviewed Oesch et al., 2007) and reconstructed skin models (Luu-The et al., 2009, Wiegand et al., 2008) were described on transcriptional level, information on their actual catalytic activities is rare.
It is known that not only parent compounds themselves but in particular their reactive metabolites possess toxic potency for instance to induce DNA damage. The objective of this study was to characterize enzymatic activities of skin models focusing on CYP-enzymes, the Flavin-dependent monooxygenases (FMO), UDP-glucuronyltransferases (UDP-GT) as well as N-acetyltransferases (NAT), which are known to be involved in toxification and detoxification processes, within two established reconstructed skin models, the epidermal EpiDerm™ (Epi-200; MatTek Corporation) and the Phenion® Full-Thickness skin model Phenion®FT (PFT, Henkel AG & Co.; Neis et al., 2010, Ackermann et al., 2010). Obtained data on metabolic activities of human reconstructed skin models could provide urgently required information to select a metabolic active skin model which is suitable for genotoxicity testing.
Section snippets
Chemicals and reagents
If not otherwise stated, all chemicals of p.a. quality were purchased from Sigma–Aldrich. Stock solutions were prepared for parabenzoic acid (PABA, 1 M DMSO), N-acetylated parabenzoic acid (PABAac, 1 mM DMSO), 4-methylumbelliferone (MUF, 5 mM DMSO), MUF-glucuronid (1 mM DMSO), 7-ethoxyresorufin (0.2 mM DMSO), pentoxyresorufin (1 mM DMSO), benzyloxyresorufin (0.5 mM DMSO), resorufin (1 mM DMSO), dithiothreitol (DTT, 100 mM in a. bidest), UDP-glucuronic acid (UDP-GA, 30 mM, DMSO), benzydamine (BA, 1 M in a.
Results
Selected enzyme activities were specified within S9 and microsomal fractions of EpiDerm™ and Phenion®FT (PFT). Table 1 summarizes obtained values from several experiments for CYPs, FMO1 and 3, UDP-GT1 and NAT1 enzyme activities. Using alkylresorufin derivatives CYP-enzyme activities remained below the LOQ and LOD of the applied methodology, the latter being specified to 0.001 [nmol/min/mg] for EROD, 0.0025 [nmol/min/mg] for PROD and 0.002 [nmol/min/mg] for BROD. Activities for Flavin-dependent
Discussion
Transcription of monooxygenases and transferases was reported within native human skin (Oesch et al., 2007) among them CYP, FMO, UDP-GT and NAT. Those xenobiotic metabolizing enzymes were selected in this study to specify their enzyme activities in two different human reconstructed skin models, the EpiDerm™ and the Phenion®FT. However, CYP-activity remained below the LOQ of the methodology for both skin models. Undetectable resorufin generation was not due to because the methodology as
Acknowledgements
This research was funded by BMBF 0315226D. We acknowledge assistance from Dr. Sibylle Gröters for histological analyses.
References (34)
- et al.
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
Toxicology in Vitro
(2010) Some distinctions between Flavin-containing and Cytochrome P450 monooxygenases
Biochemical and Biophysical Research Communications
(2005)- et al.
Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types
Toxicology in Vitro
(1997) - et al.
Xenobiotic metabolism gene expression in the EpiDerm in vitro 3D human epidermis model compared to human skin
Toxicol In Vitro
(2010) - et al.
Quantification and cellular localization of expression in human skin of genes encoding Flavin-containing monooxygenases and Cytochromes P450
Biochemical Pharmacology
(2001) - et al.
Ultraviolet-B exposure of human skin induces Cytochromes P450 1A1 and 1B1
Journal of Investigative Dermatology
(2000) - et al.
International validation of an in vitro skin irritation test protocol (EpiDerm-SIT) to replace the in vivo rabbit test for hazard identification of chemicals
Toxicology Letters
(2009) - et al.
Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin (TM) and full thickness model from Episkin (TM)
Journal of Steroid Biochemistry and Molecular Biology
(2009) - et al.
Distribution of xenobiotic metabolizing enzymes in skin
Toxicology in Vitro
(1994) - et al.
Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug
International Journal of Radiation Oncology Biology Physics
(1998)
Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of Benzydamine
European Journal of Pharmaceutical Sciences
Enzymatic and immunohistochemical studies on the role of Cytochrome-P450 and the Flavin-containing monooxygenase of mouse skin in the metabolism of pesticides and other xenobiotics
Pesticide Biochemistry and Physiology
The Phenion (R) Full-Thickness Skin Model for percutaneous absorption testing
Skin Pharmacology and Physiology
Xenobiotic metabolizing capabilities of the EpiDerm in vitro human skin equivalent: utility for assessing dermal biotransformation of pharmaceuticals and environmental chemicals
Journal of Investigative Dermatology
N-Acetyltransferase 1 enzyme activities in subtypes of HaCaT cells compared to primary keratinocytes
Naunyn-Schmiedebergs Archives of Pharmacology
Oligodeoxynucleotide uptake in in vitro cultured HaCaT keratinocytes and in a full-thickness skin model
Journal of Investigative Dermatology
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