Optimisation of a 3D living skin equivalent model for studying drug metabolism

SALLEM, Hatem Ali H. (2022). Optimisation of a 3D living skin equivalent model for studying drug metabolism. Doctoral, Sheffield Hallam University.

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Abstract

Skin is the largest organ in the human body and is used as a defence against foreign chemicals “xenobiotics”. The human skin has the capacity to metabolise foreign chemicals “xenobiotics” that pass through the stratum corneum, but knowledge of the enzymes involved is incomplete. There is an important need for validated 3D skin models, which can be utilised in the safety assessment of chemicals. An important component of the validation of these Labskin model is characterising the levels of xenobiotic metabolising enzymes (XME), particularly those that have been reported as important in the skin, Phase I XME (e.g. cytochrome P450s and flavin containing monooxygenase FMOs) and Phase II XME (e.g. glutathione S-transferase) isoforms. RT-qPCR and proteomics approach such as LC-MS/MS, nano-LC-MS/MS and DESI-MSI techniques were used to describe the profile of XME present in human skin and in vitro Labskin model that has been developed for the purpose of safety assessment. Overall, the general aims of this study were to measure the levels of Phase I and Phase II XMEs in a commercial 3D living skin equivalent model “Labskin” to determine if it is possible to recover drug response by induced genes and proteins expression and provide a 3D skin model for future drug development. The results showed that about 84 genes expression by RT-qPCR and 327 function protein by LC-MS/MS and nano-LC-MS/MS of phase I and Phase II levels were measured in the 3D Labskin model compared with native human skin. Though the expression of Phase I and Phase II level were low in the 3D Labskin model as supplied, significant induction of cytochrome P450-dependent monooxygenase (CYP1A1, CYP3A5, CYP1B1) and non-CYPs (FMO1/3) were observed after treatment with β-naphthoflavone, all-trans retinoic acid and phenobarbital which yielded a upregulated of fold change in the induced 3D Labskin model compared with 3D labskin model control. Of the enzymes indicated as present, only CYP3A5 protein was confirmed to be expressed in the 3D skin model and human skin by Western blotting. These findings suggest that the XMEs of Labskin model appears to be representative of native human skin for metabolism studies of cutaneous exposures to xenobiotics. The biotransformation of benzydamine in in vitro skin model was investigated. DESI-MS on Synapt XS and MRT was applied to evaluate the metabolic activity of FMO1/3 and CYP P450 gene/protein in 3D Labskin model using benzydamine substrate-based mass spectrometry imaging. DESI-MS detected benzydamine-N-oxide and nor-benzydamine to induced with β-naphthoflavone by FMO1/3 and P450 activity in the epidermal layer of Labskin.

Item Type:	Thesis (Doctoral)
Contributors:	Clench, Malcolm [0000-0002-0798-831X] Duckett, Catherine [0000-0002-6845-1890] Carolan, Vikki [0000-0001-7384-4018]
Additional Information:	Director of studies: Prof. Malcolm R. Clench / Supervisors: Dr. Catherine J Duckett and Dr. Vikki A. Carolan. "No PQ harvesting"
Research Institute, Centre or Group - Does NOT include content added after October 2018:	Sheffield Hallam Doctoral Theses
Identification Number:	https://doi.org/10.7190/shu-thesis-00515
Depositing User:	Colin Knott
Date Deposited:	02 Jun 2023 16:04
Last Modified:	11 Oct 2023 14:30
URI:	https://shura.shu.ac.uk/id/eprint/31970

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