Differential effects of the incorporation of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) on the binding of the transcription factors, AP-1 and TFIID, to their cognate target DNA sequences.

The thymidine analog, 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo. To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was approximately 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by MseI.

An evaluation of 6 chromosomal mutagens in the AS52/XPRT mutation assay utilizing suspension culture and soft agar cloning.

The AS52/XPRT mutation assay was examined for sensitivity in the detection of chromosomal mutagens. 6 compounds identified as chromosomal mutagens in the mouse lymphoma assay (MLA) were tested for mutagenicity in AS52 cells using suspension treatment and soft agar cloning. The 6 compounds were benzo[a]pyrene (BAP), 2-acetylaminofluorene (2AAF), methylmethanesulfonate (MMS), methyl acrylate (MCR), benzoin (BZ), and p-aminophenol (AM). BAP, 2AAF and MMS were mutagenic. The mutagenic responses for BAP, 2AAF and MMS included small colony mutants which have been shown to correlate with chromosomal mutation in the MLA. Colonies ranged from approximately 0.2 to 1.5 mm in size. The mutant frequency (MF) for the AS52 cells treated with BAP and 2AAF exceeded that previously reported for the MLA by 2-fold. In contrast, the MF for AS52 cells treated with MMS was one-third that reported in the MLA. The MF obtained in AS52 cells exceeded that reported for the CHO/HGPRT mutation assay for all 3 compounds. MCR, which produces almost entirely small colonies in the MLA, was negative in AS52 cells as were the MLA chromosomal mutagens AM and BZ. However, AM and BZ have only been reported mutagenic in the MLA. Both are considered nongenotoxic and noncarcinogenic. The results with the latter 3 compounds suggest that the AS52 assay is not as sensitive as the MLA for the detection of compounds identified as chromosomal mutagens.

The evaluation of methapyrilene for bacterial mutation with metabolic activation by Aroclor-induced, methapyrilene-induced and noninduced rat-liver S9.

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.1% methapyrilene in the diet for 10 weeks prior to sacrifice. S9 was prepared from the livers of the control, MP-treated and Aroclor-induced Fischer 344 rats. Each type of S9 was analyzed for mixed function oxidase activity, cytochrome P-450, and protein content. MP was then evaluated for mutagenicity in 6 strains of S. typhimurium (TA1535, TA1537, TA98, TA100, TA2638 and TA104) and one strain of E. coli (WP2uvrA-) using the standard plate-incorporation assay. MP was not mutagenic in any of the 7 bacterial strains when tested at concentrations < or = 10 mg/ml in the presence of each type of S9. However, in the absence of metabolic activation, an approximate 2-fold increase in revertants was noted with strain TA1535. The data from this study show that MP was not converted to a mutagenic metabolite by any of the three S9 types examined. However, the "weak" positive response with strain 1535 in the absence of metabolic activation indicates that further research is needed to elucidate the mechanism of action of this rat carcinogen.