Elsevier

Toxicology in Vitro

Volume 25, Issue 6, September 2011, Pages 1209-1214
Toxicology in Vitro

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

https://doi.org/10.1016/j.tiv.2011.03.012Get rights and content

Abstract

With the perspective to use human reconstructed skin models for genotoxicity testing which require metabolic activation of xenobiotics, this study aimed to characterize activities of biotransforming enzymes within two human reconstructed skin models, the epidermis model EpiDerm™ (MatTek) and the Phenion® Full-Thickness skin model Phenion®FT (Henkel). According to existing gene expression profiles, Cytochrome P450 (CYP) enzymes, Flavin-dependent monooxygenases (FMO), N-acetyltransferases (NAT) and UDP-glucuronyltransferases (UDP-GT) were investigated in S9 or microsomal fractions. CYP-catalyzed monooxygenation was assayed using 7-ethoxyresorufin, pentoxyresorufin and benzyloxyresorufin as substrates. FMO activity was tested using benzydamine. Conjugating activities of NAT and UDP-GT were determined by acetylation of p-aminobenzoic acid or glucuronation of 4-methylumbelliferone, respectively. Although CYPs were detected by expression profiling, no CYP activity was detected in either the epidermal nor the full-thickness reconstructed skin model while expression and activity of FMO, UDP-GT and NAT were demonstrated in both.

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.

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