Label-free Quantitative Proteomics and Substrate Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in ex Vivo Human Skin and a Human Living Skin Equivalent Model

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  • External authors:
  • Narciso Couto
  • Jillian R.A. Newton
  • Cristina Russo
  • Esther Karunakaran
  • James Sidaway
  • Malcolm R. Clench

Abstract

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases and nucleases in six human skin explants and a 3D living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C (ADH1C), the most abundant phase I XME in human skin, and glutathione S-transferase pi 1 (GSTP1), the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (CYP) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate CYPs were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin (TXN) and peroxiredoxin-1 (PRDX1). This systematic determination of functional equivalence between human skin and LabSkin is a key step towards the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs.

Bibliographical metadata

Original languageEnglish
JournalDrug Metabolism and Disposition
Early online date2 Nov 2020
DOIs
Publication statusE-pub ahead of print - 2 Nov 2020

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