DNA adduction in context: Native metabolic activation of a foodborne procarcinogen and adducted oligonucleotide sequencing monitored by LC-MS/MS.

摘要

DNA adducts are purported to be useful biomarkers of environmental exposure to a variety of agents. On a daily basis, potential procarcinogenic and carcinogenic exposures from food, the environment and endogenous routes serve to create a burden of exposure that over 20 to 30 years ultimately leads to cumulative genetic damage and cancer. In order to appreciate the magnitude of the genotoxic exposure problem relating to DNA damage, Chapter 1 provides a review of one class of procarcinogens related to foodborne exposure: heterocyclic aromatic amines. Their formation, metabolism and quantitation are detailed in order to establish the basis for evaluating this class of compounds in this thesis.;Having established the importance of testing this class of compounds, Chapter 2 details the development of a cell-based method for the native metabolic activation of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenyl-imidazo[4,5-beta]pyridine (PhIP). The metabolically competent human lymphoblast cells (MCL-5) are recombinantly engineered to contain 5 Cytochrome P450 enzymes capable of Phase I redox metabolism. Dosing studies involving variable concentrations of PhIP from 5-40 muM incubated over 24 hours, a 10 muM dosing of PhIP incubated over 36 hours and a 5 muM dosing of PhIP for 6 weeks were done to evaluate the formation of PhIP-dG DNA adducts under normal cell growth conditions. An LC-MS/MS method was developed and validated for the quantitation of PhIP-dG adducts isolated from the MCL-5 cell DNA using a unique nanoelectrospray LC source.;Chapters 3 and 4 continue the theme of this thesis by evaluating the sequence specific context of DNA adduction within oligonucleotides. In these chapters, methodology was developed to establish the appropriate mobile phase, column stationary phase and nanospray emitter type in order to analyze adducted oligonucleotides on a LCQ Classic ion trap. Because previous literature reports utilized an ion pairing reagent that was detrimental to the LCQ Classic, new LC conditions were required. Chapter 4 in this thesis used the LC-MS/MS method developed in Chapter 3 for the analysis of BPDE adducts formed in a 14-mer oligonucleotide sequence in which CpG dinucleotide sites were varied from monomethylated primary strands to permethylated oligomers. Adduct quantity as a function of adduct position in the 14-mer primary strand was determined without the need for digestion of the oligonucleotide.;Chapter 5 evaluates the future direction of DNA adduct work for PhIP in which the quantity of PhIP-dG DNA adduct is related to gene expression changes in a bronchial epithelial cell line. Serial dilutions studies show a distinct concentration at which no transcriptional effects are observed as well as no DNA adducts detected. Additional recommendations are provided for the expansion of the use of monolithic columns for several challenging oligonucleotide problems.