Mutagenic Specificity of N-Nitrosotaurocholic Acid in supF Shuttle Vector Plasmids

The mutagenic speciˆcity of N-nitrosotaurocholic acid (NO-TCA) in human cells was investigated using supF shuttle vector plasmids.The plasmids pMY189 were treated with NO-TCA in vitro and introduced into normal ˆbroblasts (WI38-VA13) and nucleotide excision repair (NER)-deˆcient cells (XP2OS(SV)) for replication.The background mutation frequency of the supF gene was 4.1× 10 -4 and 2.0×10 -4 after replication in normal and NER-deˆcient cells, respectively.The mutation frequency increased 5 and 15 times in normal and NER-deˆcient cells, respectively, after the treatment of the plasmid with 50 mg/mL of NO-TCA.The higher mutation frequency in NER-deˆcient cells indicates that the DNA damage induced by NO-TCA is repaired by NER.Base sequence analysis of 101 and 94 plasmids with mutations in the supF gene propagated in normal and NER-deˆcient cells, respectively, revealed that the majority of the mutations were base substitutions (about 89 and 90%) and the rest were deletions and insertions (about 11 and 10%) in both cell lines.About half of the mutant plasmids contained a single base substitution.Of the single base substitutions, the most frequent mutations were G:C to A:T transitions (about 37 and 36%), followed by G:C to C:G transversions (about 31 and 28%) in both cell lines.The mutations were not distributed randomly but were located at several hot spots in the supF gene, and almost all hot spots were at G:C sites.These observations accord with previous ˆndings that NO-TCA forms DNA adducts with dC and induces G:C to A:T base substitution in Salmonella typhimurium TA100.