Date of Award
Doctor of Philosophy (PhD)
The nitro substituted polycyclic aromatic hydrocarbons (nitro-PAH) are a class of environmental contaminants which are potent mutagens in a number of biological test systems. The three dinitropyrene (DNP) isomers are of special interest because of their extremely high mutagenicity in the Ames Salmonella typhimurium reversion assay. Metabolic studies in S. typhimurium using both tritium labelled and unlabelled DNP demonstrated that the DNPs undergo nitro reduction and N-acetylation. Mutagenicity studies of DNP metabolites in S. typhimurium strains demonstrated that the reactive metabolite responsible for the mutagenic activity of the DNPs is at the oxidation state intermediate between nitroso-nitropyrene and amino-nitropyrene and both nitro reduction and acetylation are required for expression of mutagenicity. Efforts were directed toward synthesis of hydroxylamino-nitropyrenes (HANP) derivatives as they appeared to be likely candidates for the reactive metabolite and could be easily synthesized. The 1,8-HANP derivation was shown to produce the same covalently DNA bound adduct as that produced in vivo by the bacterial metabolism of 1,8-DNP. The 1,3-and 1,6-substituted HANP derivatives were also shown to produce covalently bound DNA adducts in vitro. Based upon the known structures of other DNA-adducts, it was postulated that these in vitro adducts were the result of bond formation between the exocyclic amino nitrogen of an amino-nitropyrene moiety and C-8 of 2'-deoxyguanosine. However, the characterization of these adducts presented many difficulties. Acquisition of UV-visible spectra of these DNP-DNA adducts was aided by coupling reverse phase high pressure liquid chromatography (RPLC) with diode array detection. The RPLC analyses of these adducts also demonstrated that they undergo extensive degradation under both acidic and basic conditions. Interpretation of their NMR spectra was difficult because of line broadening effects due to stacking of the adducts in solution. The acquisition of mass spectra of these in vitro was difficult because of their low volatility and pH instability. A number of "soft" ionization techniques such as "in-beam" ionization, fast atom bombardment (FAB), field desorption (FD), and coupled LCFAB-MS were evaluated, but only poor quality spectra were obtained. These difficulties were overcome by producing the trimethylsilyl and t-butyldimethylsilyl derivatives MS analysis of these derivatives using desorption electron ionization (DEI) mass spectrometry produced mass spectra that were rich in fragmentation ions allowing final characterization of the adducts. Finally the interaction between calf thymus-DNA and aminonitropyrene (ANP) or diaminopyrene (DAP) metabolites was studied because these metabolites were structurally similar to known DNA intercalators. The interaction of these metabolites was shown to be different than the structurally similar intercalators.
Andrews, Paul, "The fate of dinitropyrenes in Salmonella typhimurium metabolism and DNA-adduct formation" (1988). Open Access Dissertations and Theses. Paper 3562.