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Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic

  • Toxicokinetics and Metabolism
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Abstract

Skin function is not limited to a physical barrier. According to its total surface area, it is also considered as an extra-hepatic metabolizing organ. In vitro engineered human skins have been developed to replace limited ex vivo normal human skin samples (NHS). Thus, assessing and comparing skin models from SkinEthic [Episkin™, RHE™ and the full thickness model (FTM)] with NHS in terms of metabolic capability are essential. The apparent activities of main cutaneous isoforms of cytochrome P450-dependent monooxygenases (CYP1A1/1B1, 2B6/2C18/2E1, 3A5/3A7), esterase, glutathione-S-[(GST), A, M, P, T], N-acetyl-(NAT1), uridinyl-diphosphate glucuronyl-(UDPGT 1A family) and sulfo-(SULT1A1) transferases were determined using probe substrates. Mean activities indicative of CYP1A1/1B1 (expressed as pmol/mg protein/6 h) in RHE™ (2.8) and FTM (2.6) were very similar to NHS (3.0) while Episkin™ showed a higher activity (9.1). Activities of CYP3A5/3A7 in FTM (3.3) and Episkin™ (3.6) were similar to NHS (3.8) while activity in RHE™ (13.3) was higher. CYP2B6/2C18/2E1 activity was below LOQ (0.5) in all skin models and NHS. Comparable intrinsic metabolic clearances were measured between NHS and skin models for esterase, UDPGT, GST and NAT1 activities. SULT1A1 activity toward probe substrates was not detected in skin models and observed at the limit of detection in NHS. Weak cytochrome P450-dependent monooxygenases, high esterase and transferase activities suggested that NHS and skin models exhibited limited functionalization and much greater detoxification (hydrolytic and conjugating) capacities. These results demonstrate that skin models are similar to NHS in terms of metabolic functionality toward xenobiotics investigated and useful tools to assess both the local efficiency and safety of cosmetics.

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Abbreviations

BSA:

Bovine serum albumin

CDNB:

Chloro-2,4-dinitrobenzene

CYP:

Cytochrome P450-dependent monooxygenases

DNPSG:

Dinitrophenyl S-glutathione conjugate

EROD:

7-ethoxy resorufin O-deethylase

ES:

Esterases

FTM:

Full thickness model of SkinEthic

GST:

Glutathione-S transferases

HPLC:

High-performance liquid chromatography

LOD:

Limit of detection

LOQ:

Limit of quantification

MTT:

1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide

NAT:

N-acetyl transferases

α-NF:

α-Naphthoflavone

NHS:

Normal human skin

PABA:

para-Aminobenzoic acid

PAcBA:

para-Acetamidobenzoic acid

PNP:

para-Nitrophenol

PNPS:

para-Nitrophenyl sulfate

RT-PCR:

Real-time polymerase chain reaction

SDS:

Sodium dodecyl sulfate

S.E.M:

Standard error of the mean

SPE:

Solid-phase extraction

SULT:

Sulfo-transferases

TCA:

Trichloroacetic acid

TFA:

Trifluoroacetic acid

UDPGT:

Uridyl diphosphate glucuronyl transferases

4-MU:

4-Methylumbelliferone

4-MUAc:

4-Methylumbelliferyl acetate

4-MUG:

4-Methylumbelliferyl-β-glucuronide

7-BFCOD:

7-Benzyloxy-4-(trifluoromethyl)-coumarin O-debenzylation

7-BFC:

7-Benzyloxy-4-(trifluoromethyl)-coumarin

7-ER:

7-Ethoxyresorufin

7-HFC:

7-Hydroxy-4-(trifluoromethyl)-coumarin

7-MFCOD:

7-Methoxy-4-(trifluoromethyl)-coumarin O-demethylase

7-MFC:

7-Methoxy-4-(trifluoromethyl)-coumarin

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Acknowledgments

We wish to thank Pr. Daniel Mansuy (UMR 8601, Paris Descartes University, France), Dr. Marcel Delaforge (URA 2096, CEA Saclay, Gif-sur-Yvette), Pr Van Luu-The (Oncology, Molecular Endocrinology and Genomic Research Center, Quebec University Hospital Research Center and Laval University, Quebec, CANADA) and Dr Bruno A. Bernard (L’Oréal Research and Innovation, France) for their advice and scientific assistance. We would also like to thank Oroxcell SA for its technical assistance in P450-dependent monooxygenase experiments.

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  1. L’Oréal Research and Innovation, 1, Av. Eugene Schueller, 93601, Aulnay sous Bois Cedex, France

    Joan Eilstein, Guillaume Léreaux, Natali Budimir, Georges Hussler & Daniel Duché

  2. Newcastle upon Tyne, NE1 7RU, UK

    Simon Wilkinson

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  1. Joan Eilstein
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  2. Guillaume Léreaux
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  3. Natali Budimir
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  4. Georges Hussler
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Correspondence to Joan Eilstein.

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Eilstein, J., Léreaux, G., Budimir, N. et al. Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic. Arch Toxicol 88, 1681–1694 (2014). https://doi.org/10.1007/s00204-014-1218-6

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  • DOI: https://doi.org/10.1007/s00204-014-1218-6

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