Biomarkers of exposure to nitrosation products of amino acids and peptides
thesisposted on 2014-12-15, 10:41 authored by Kathryn Lisa. Harrison
The endogenous nitrosation of dietary amino acids and peptides may be a major source of genotoxic damage in the stomach and lower GI tract. Amongst the many N-alkyl-N-nitrosocompounds and related compounds, there are a number which share the common features of being derived from glycine and are carboxymethylating agents.;Novel immunoaffinity-RP-HPLC-fluorescence methods were developed allowing the concomitant detection of O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) with limits of detection of 0.128mol O6-CMdG/mol dG and 0.064mol O6MedG/mol dG in lmg of DNA. N-(N'-acetyl-L-prolyl)-N-nitrosoglycine (APNG), azaserine and potassium diazoacetate (KDA) all reacted with calf thymus DNA in vitro to give the expected O6-CMdG as well as lesser amounts of O6-MedG. The ratios of O6-MedG to O6-CMdG were 1:9.73 (APNG), 1:16.12. (KDA) and 1:38.45 (azaserine). However, these nitrosated glycine derivatives showed large differences in their capacity to alkylate DNA with O6-alkylation formation ratios of 1:12.1:137.9 for 5mM azaserine, APNG and KDA respectively.;As O6-CMdG is not repaired by O6-alkylguanine alkyl transferases and is likely to be persistent in mammalian tissues, a sensitive immunoslotblot assay (0.32mol O6-CMdG/mol dG) which required only 1g DNA for O6-CMdG was developed. O6-CMdG was detected in the stomach DNA of experimental animals treated with KDA or APNG by gastric intubation. Interestingly, control animals and non-target tissues of treated animals (liver, intestine) also had detectable levels of O6-CMdG.;Examination of the DNA from human gastric biopsies from 30 individuals involved in a H. pylori study, revealed detectable amounts of O6-CMdG in 27 samples ranging from 0.60-19.79mol/mol dG. No statistically significant difference was found between individuals with or without H. pylori infection on the basis of O6-CMdG levels, although the highest levels observed were all in infected individuals.
Date of award1998-01-01
Awarding institutionUniversity of Leicester