IN-VIVO MUTAGENESIS OF THE REPORTER PLASMID PSP189 INDUCED BY EXPOSURE OF HOST AD293 CELLS TO ACTIVATED POLYMORPHONUCLEAR LEUKOCYTES

We measured the mutation frequency and spectrum induced by exposure of the mutation reporter plasmid pSP189 in vivo to phorbol myristate ace tate (PMA)-activated human polymorphonuclear leukocytes (PMNs). The mu tation frequency induced in the supF tRNA gene of pSP189 transfected i nto human Ad293 cells by a 30 min exposure to 4 x 10(6) activated PMNs /ml was 3- to 9-fold higher than the background mutation frequency of 0.1-1.8 x 10(-5). The enhanced mutation frequency caused by activated PMNs required replication of the reporter plasmid in host Ad293 cells, Fifty five unique activated PMN-associated mutants characterized by s equencing included base substitutions (55%) and deletions (45%), howev er, no small (1-3 bp) deletions were observed, Ninety four percent of point mutations occurred at C:G base pairs, with C:G-->T:A transitions (47%) and C:G-->A:T transversions (37%) predominating, A prominent ho t-spot was observed at d(pCAGAC) on the tRNA strand, Although H2O2 gen eration was required for mutagenesis, the mutation spectrum induced in pSP189 by in vivo exposure to activated PMNs differed from that induc ed by in vivo exposure to H2O2, It also differed from the spectrum ind uced in single-stranded DNA in vitro by activated PMNs, suggesting tha t the mutational spectrum is a complex function of the kinetics of rea ctive oxygen generation and factors contributed by the target cell.