Kinds of mutations formed when a shuttle vector containing adducts of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene replicates in human cells.

Abstract

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) replicates in human cells. A human embryonic kidney cell line, 293, was used as the eukaryotic host. The target gene for mutation analysis, supF, codes for a tyrosine suppressor tRNA and is strategically located between the origin of replication of the plasmid in Escherichia coli and the gene for a selectable marker, so that the possibility of recovering supF mutants containing gross rearrangements is low. The frequency of supF mutants obtained when untreated plasmid replicated in 293 cells was 1.4 X 10(-4). The frequency with BPDE-treated plasmid increased linearly as a function of the number of adducts, with 16 adducts per plasmid giving 38 X 10(-4). Polyacrylamide gel and agarose gel electrophoresis analysis of 137 plasmids with mutations in the supF gene indicated that 70% (21/30) from untreated plasmids contained deletions or insertions or showed altered gel mobility, whereas only 28% (30/107) of those derived from BPDE-treated plasmids contained such alterations. Of the 86 unequivocally independent mutants derived from BPDE-treated plasmids that were analyzed by sequencing, the majority (60/86) exhibited base substitutions. Mutants exhibiting frameshifts (insertions or deletions of one, two, or four base pairs) were also found, but they were a minority (11/86). In the progeny of BPDE-treated plasmids 61/71 base substitutions observed were transversions, with 45/61 G X C----T X A. Examination of the location of BPDE-induced mutations among the 85 base pairs in the structure of the tRNA revealed that 30% of the base substitutions occurred at two sites and 44% of the rest occurred at five other hot spots. Only 20% of all these base changes involved a site in which a guanine containing a BPDE adduct is predicted to be labile--i.e., a guanine that has a pyrimidine to its 5' side.