The mutagenic spectrum of acridine-linked aniline nitrogen mustards in AS52 cells: implications of DNA targeting with high selectivity for adenine or guanine bases

Abstract

The mutational spectra generated in AS52 cells at the gpt gene locus by aniline mustards were studied by the isolation of resistant clones and sequencing of the altered gene. A set of four aniline mustards (both mono- and bifunctional) linked to a DNA-affinic 9-aminoacridine (9-AA) carrier was used, together with the untargeted mustards chlorambucil (CHL) and its half-mustard, and the DNA binding carrier, 9-AA. Both 9-AA and CHL were weak cytotoxins, with the DNA-targeted mustards being markedly (10–40-fold) more dose potent, and the bifunctional ones somewhat more toxic than the monofunctional ones. 9-AA produced a different spectrum of mutations to the spontaneous background, with more minor addition events and less base pair substitutions, and showing for the first time that frameshift events so characteristic of 9-AA in bacteria or bacteriophage also occur in mammalian cells. The mutational spectra of the DNA-targeted mustards were quite different both from this and from the lesions caused by the untargeted mustards, which cause largely transition mutations at AT sites (despite a clear preference for formation of N 7-guanine adducts). There were very few transition mutations, suggesting that the initial O 6-alkylguanine/ O 4-alkylthymine lesions considered to give rise to these are relatively rare. There was also a lower incidence of complete deletions, usually attributed to DNA cross-links. For the short chain length targeted mustards, which form initial stable adducts largely (95%) at guanine N 7 sites, base pair substitution mutations, predominantly transversions, involved AT and GC base pairs equally. In contrast, the longer chain length targeted mustards, which form >90% of initial adducts at adenine N 1 sites, generated also formed transversion mutations, but these overwhelmingly (24/27) involved AT base pairs.