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

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.

Mass spectrometry‐based abundance atlas of ABC transporters in human liver, gut, kidney, brain and skin

“…1A). In addition, ABCC12, found only in skin samples, was actually close to the limit of detection and not found if alternative processing parameters were applied [38]. The Venn diagram in Fig.…”

“…Human adult livers (Adult Liver Set 2, n = 39), removed from histologically normal tissue adjacent to tumours, were obtained from the Manchester Royal Infirmary, Manchester University NHS Foundation Trust, UK, with ethical approval for use in research from North West Research Ethics Committee, UK (14/NW/1260, 19/NW/0644 [37]. Skin tissues ( n = 6) were obtained from individuals undergoing routine abdominoplasty surgery by the Teaching Hospital of the University of Bradford; ethical approval for use in research was given by the Independent Ethics Committee (36‐DRMBPY‐06‐001) [38]. In addition, two living skin equivalent (LSE) models were supplied by Labskin UK Ltd (York, UK); these were delivered after 14 days of development in transport culture medium.…”

“…Liver and kidney samples were processed to microsomes, whereas brain and intestine tissues were processed to microvessels and mucosal fractions, respectively. For skin, S9 fraction (postmitochondrial fraction) was prepared [38]. Briefly, the subcutaneous layers were removed before skin samples were further processed.…”

“…Digestion protocols were based upon endopeptidase Lys‐C, followed by trypsin. Crude membrane proteins were spiked with stable isotope‐labelled MetCAT and/or TransCAT [40] in the case of Adult Liver Set 2, intestine, kidney and brain microvessel samples (these are examples of Quantification conCATemer (QconCATs), peptide standards concatenated to form a single artificial protein) or with a mixture of exogenous protein standards at known concentrations in the case of paediatric livers, Adult Liver Set 1 [15] and skin samples [38]. The external standards used in the latter set consisted of BSA, horse myoglobin and yeast aldehyde dehydrogenase (ADH).…”

“…In addition, two living skin equivalent (LSE) models were supplied by Labskin UK Ltd (York, UK); these were delivered after 14 days of development in transport culture medium. LSE is treated as a single skin sample in this report, but are described in more detail elsewhere [38]. Paediatric Liver Set 1 was made up of samples ( n = 8) provided by Invitron (Monmouth, UK) and samples ( n = 12) obtained as microsomes from XenoTech (Lenexa, KS, USA).…”