Activated polymorphonuclear leukocytes (PMNs) have been shown to generate reactive oxidizing and free radical species which cause DNA damage, mutation, and neoplastic transformation. Activated PMNs are a biologically significant model with which to characterize the damage to DNA caused in vivo by free radical and oxidant attack. We propose to characterize in vivo DNA base damage in human cells caused by activated PMNs using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS/SIM) to quantify base modifications, and using the ligation-mediated polymerase chain reaction technique (LMPCR) to map DNA base damage at the nucleotide level. GC-MS/SIM is a sensitive, broad-spectrum analytic technique, capable of quantifying the ten principal base modifications caused by reactive oxygen species under physiologic conditions. We will use this technique to obtain a global assessment of activated PMN-induced in vivo base damage. LMPCR is a technique which maps and quantifies base damage at individual nucleotides by cleaving DNA preferentially at damage sites; resultant single strands are visualized by converting them to double strands, ligating common linkers, amplifying with an appropriate set of primers via the polymerase chain reaction, separating bands by polyacrylamide gel electrophoresis, blotting to a membrane and radioactively probing. Each band corresponds to a particular nucleotide at which damage occurred; band intensities correspond to damage frequency. In this proposal, we will map activated PMN-induced base damage in human cells which is sensitive to cleavage by hot piperidine; we will also use enzymes to adapt the technique to map abasic sites and guanine-derived lesions, including 8-oxoGua. Base damage will be mapped in SupF, an episomal gene used in our mutation studies, and the genomic gene pgk 1; factors which influence the distribution of activated PMN-induced base damage in vivo will be assessed. We will also determine the mutation frequency generated by in vivo exposure of human Ad293 cells to activated PMNs, using the mutation reporter plasmid pSl89. pSl89 carries the suppressor tRNA gene SupF; mutations in SupF are identified by recovering the plasmid and transforming the bacterial reporter strain MBM7070, which carries a SupF-suppressible amber mutation in its lacZ gene. Individual mutations will be characterized by standard dideoxy sequencing, in order to develop a mutation spectrum which can be compared to the LMPCR-derived base damage map. These data will help elucidate the mechanism(s) by which exposure of human cells to a flux of free radical and oxidizing species causes mutation, an important step in carcinogenesis.
