Evaluating skin xenobiotic metabolism using a tissue engineering and proteomics approach for drug induced toxicity

Background: Xenobiotics are foreign molecules that interact with our cells. The presence of metabolising enzymes in skin is of considerable importance for the pharmaceutical/cosmetic industry as xenobiotic compounds delivered either topically or systemically may be metabolised within the skin to produce active metabolites that may cause toxicity or hypersensitivity leading to tissue damage. However, questions remain regarding metabolising enzyme kinetics, identification and quantification of metabolites and their rates of elimination from the skin.
Hypothesis: Using a combined tissue engineering, proteomic and metabolomic approach can determine key metabolism pathways involved in skin toxicity and hypersensitivity.
Specific Aims:
Generate immuno-competent tissue engineered skin equivalents
Investigate spatial location of key phase 1 and phase 2 enzymes in tissue models
Identify parent compounds and metabolites within the skin and their subsequent clearance from the tissue
Determine if these metabolites cause a hypersensitivity reaction
Elucidate key pathways and proteins involved in skin toxicity and hypersensitivity
To provide knowledge transfer with systems biologists to add prediction profiling
Research Plan:Tissue engineered skin equivalents will be constructed from skin keratinocytes and dermal fibroblasts and cultured at an air-to-liquid interface to produce a 3D model containing a multi-layered, stratified squamous epithelium. These models are routinely made in our laboratory (primary supervisor, University). Skin models will be developed further to incorporate dendritic (Langerhans) cells as these are key mediators of tissue hypersensitivity. Expression and spatial location of xenobiotic enzymes within the engineered tissue will be examined by qPCR, immunoblotting, immunohistochemistry and mass spectrometry imaging and validated against native excised human skin. To examine xenobiotic metabolism, molecules with known metabolites will be incubated with skin equivalents and their metabolites measured using mass spectrometry (LCMS) over time (secondary supervisor, MRC Associate Partner RO). In addition, xenobiotic compounds with known metabolites that instigate a hypersensitivity reaction will be added to skin equivalents and the activation and movement of dendritic cells out of the epithelium will be monitored using imaging techniques (confocal microscopy) (primary supervisor, University). Analysis of key pathway involved in toxicity/ hypersensitivity will then be investigated by global proteomics and phosphoproteomics. This project will provide the student with expertise in tissue-engineering, proteomics and metabolomics. The data generated from this project will also feed directly into an existing collaboration on Mathematical/Systems Modelling of xenobiotic metabolism and metabolite production in skin (third supervisor, Liverpool John Moores University). The experimental data will inform the development of a new mechanistic-based mathematical modelling system that will ultimately be able to predict the quantities and location of the metabolites produced from any given xenobiotic compound within the skin to cause toxicity/hypersensitivity reactions. The student will have the opportunity to validate the mathematical models using the newly created in vitro model. This project offers the student an opportunity to contribute to an established multidisciplinary team that involves scientists, mathematicians and clinicians working within the Northern Health Sciences Alliance to improve drug safety.