Brookman-Amissah, Nicola; (2000) Identification of a novel cytochrome P450 cDNA in rat oesophagus: Relevance to carcinogenic N-nitrosamine metabolism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oesophageal cancer is an invariably fatal form of cancer with about 7000 deaths per annum in the UK alone. Evidence from the epidemiology and the mutation spectrum in the p53 gene suggests that the more common form of this cancer, squamous cell carcinoma, is caused by exposure to carcinogens. The N-nitrosamines are candidate carcinogens for this cancer in man. N-nitrosamines are metabolically activated by cytochromes P450 (P450s) and their organotropism is largely dependent on the distribution in the body of the particular nitrosamine and the P450s capable of metabolising it. The rat oesophagus is particularly susceptible to methylation and tumour formation by asymmetric N-nitrosamines, many of which selectively induce oesophageal tumours. This suggests that the oesophagus may contain a P450 that is absent or rare in other organs. The nature of this nitrosamine-metabolising P450 was investigated. A combination of Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Rapid Amplification of Cohesive Ends-Polymerase Chain Reaction (RACE-PCR) and oesophageal cDNA library screening showed the expression in the rat oesophagus of a novel P450 of the 2B subfamily. The deduced amino acid sequence of this P450 shows 84%, 83% and 77% identity to those of CYP2B1, CYP2B2 and CYP2B12 respectively. The catalytic activity of this new P450 is not yet known but metabolism and methylation studies in vivo using rats treated with phenobarbital, a CYP2B1/2B2 inducer, confirmed that these members of the 2B subfamily can metabolise the oesophagus selective nitrosamine, N-nitrosomethyl-n-butylamine, and that a major part of this metabolism was activating hydroxylation of the a-carbon of the butyl group. This supports the view that the novel P450 identified in rat oesophagus may be responsible for the metabolic activation and carcinogenicity of nitrosamines in the oesophagus. Further in vivo metabolic studies of N-nitrosomethyl-n-butylamine using untreated rats showed that it is metabolised in rat liver by CYP2E1. However, in this case 75% of the metabolism is detoxifying. An increase in methylation of oesophageal DNA by the nitrosamine after ethanol administration showed that CYP2E1 is not involved in the oesophageal metabolism of this nitrosamine.

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