Daniel James Hitchcock

Background

Understanding responses to the exposure of xenobiotic substances is a fundamental aspect of toxicology. Scientists study the impact of toxicological compounds at different levels, from ecosystems right down to small biomolecules. In addition, many organisms have mechanisms which deal with the detoxification of foreign compounds, and it is here that we wish to investigate the ability for organisms to respond to various chemicals. A certain type of nuclear receptor, known as the promiscuous xenobiotic receptor (PXR), is a receptor found in many organisms which is important in the regulation and detoxification of potentially toxic compounds. However, while many of these compounds have the ability to activate transcription factors from this receptor, many others can antagonise its action. In addition, different chemicals affect species in completely different ways. There are numerous ways to assess the mode of various chemicals in this research project and we hope to find strengthen current methods and explore new ones in order to assess this.

Aim

This project will assess how PXR responds to various xenobiotic substances, across a range of species. Previous studies have investigated the receptor’s ability to regulate the expression of target genes in certain types of zebrafish, humans, mice and land mammals, but few have dealt with many species found within or near Norway such as cod, seabirds and seals. This study hopes to investigate PXR action through different assays and determine whether the methods correlate or show any possible species differences that might occur. 

Method

This project hopes to assess xenobiotic action of PXR by cloning mRNA orthologues obtained from the liver of various species and carrying the ligand binding region of a yeast transcription factor, Gal4, into a plasmid. These plasmids will be used in a luciferase reporter assay, whereby plasmids are seeded in simian Cos-7 kidney cells, transfected with luciferase reporter plasmids, the ligand of interest is added and the activation of the PXR is determined by measuring the absorbance of the genes in plates after their exposure to a controlled concentration of luciferase. In addition, we can expose PXR to each ligand of varying concentration to determine whether an optimal level of expression may occur.

We also wish to test other methods such as using the AlphaLISA assay and identifying monoclonal antibodies which can be useful in the monitoring of PXR activation.

Supervisor: Anders Goksøyr
Assistant supervisor: Roger Lille-Langøy

References

Milnes, M.R., Garcia, A., Grossman, E., Grün, F., Shiotsugu, J., Tabb, M.M., Kawashima, Y., Katsu, Y., Watanabe, H., Iguchi, T. and Blumberg, B. 2008. Activation of Steroid and Xenobiotic Receptor (SXR, NR1I2) and Its Orthologs in Laboratory, Toxicologic, and Genome Model Species. Environmental Health Perspectives 116: 880–885.