Abstract
Long term exposure to peroxisome proliferating compounds in rats, mice and rabbits lead to the onset of hepatocarcinogenesis by non-genotoxic mechanisms. This onset of this disease is linked to the induction of enzymes from the Cytochrome P4504A (CYP4A) sub-family. This induction has been attributed to an increase in transcription of the genes for these enzymes. The aim of this thesis was to compare the transcriptional regulation of the rat and human CYP4A genes in order to predict the susceptibility of man to the same peroxisomal proliferation seen in the rat and other responsive species. In order to undertake this project it was necessary to isolate the rat CYP4A genes with their upstream regulatory regions and their human analogues. Once isolated the promoter regions of the gene could be analysed in the hope that the elements in the rat gene that made it susceptible to induction by peroxisome proliferation could be identified and searched for in the human sequence. Transcriptional activation assays could also be performed as a further indication of the inducibility of the human gene by peroxisome proliferators. In this Thesis I report on the following results: 1) We succeeded in isolating a gene (including its 5' regulatory region) from the human CYP4A family which had strong homology in exonic regions with the CYP4A11 cDNA sequence published by Palmer et al [1993b]. The gene was sub-cloned into manageable sections and inserted into plasmid vectors for ease of handling. The clones were partially sequenced and good consensus contigs obtained for 9.5kb of the isolated gene. From the contig sequences a comparison of the human gene to other members of the CYP4A family was performed and it was determined that, although the whole of the coding region had not been sequenced, the gene belonged to the CYP4A family. 2) Using part of the cloned gene in genomic Southern blotting analysis we determined that the multiplicity of CYP4A forms seen in the rat and rabbit is not seen in humans. While we could not rule out the presence of very closely related isoforms, the weight of evidence points to a single CYP4A form in man. 3) Limited analysis on the role of PPAR in the regulation of this gene has proved inconclusive. The gene does share several regulatory motifs with other members of the CYP4A family and more sequencing of the promoter region may complete the picture.