Abstract 2731: Detection of blastomogenic activity of DNA minor groove binding ligands by SMART in Drosophila

Abstract

Abstract The increasing request of chemical safety assessment demands alternative methods to animal experimentation. Internationally accepted short-term assays are capable of detecting five different categories of change: gene mutation; clastogenicity; aneuploidy and non-disjunction; DNA damage as measured by DNA repair; in vitro cell transformation. Recently it was demonstrated that loss of heterozigocity (LOH) through homologous recombination, causing manifestation of a recessive mutant allele, represent frequent DNA rearrangements in tumors of patients with inherited retinoblastoma and Li-Fraumeni syndrome. LOH may be detected easily using the test for somatic mutation and recombination in Drosophila elaborated more than 30 years ago in our laboratory and currently known as SMART. The assay is based on the manifestation of recessive mutant allele after inactivation of wild type one due to somatic point mutation, chromosomal deletion or loss of heterozygocity. Recently we improve SMART to detect blastomogenic activity of chemicals using wtsP4/+ heterozygous larvae. Wts gene is involved in cell cycle regulation and its homologues are tumor suppressor genes in mice and human. Hereby we demonstrate benefits of SMART in the analysis of genotoxic effects of minor groove binding ligands (MGBLs), Hoechst 33258 and Hoechst 33342, and two new bisbenzimidazols: 1-[6-(4-methylpiperazin-1-yl)-1H,3’H-2,5’-bibenzimidazol-2’-yl]methanacetamide N-[6-(4-methylpiperazin-1-yl)-1H,3’H-2,5’-bibenzimidazol-2’-yl]methanacetamide. Hoechst 33258 and Hoechst 33342 are widely used in molecular biology. Evaluation of their genotoxicity is complex due to the mode of its action. They neither alkylate DNA nor form interstrand-crosslinks or DNA adducts and bind DNA via electrostatic, hydrogen and van der Waals bonds and inhibit some enzymes, causing genetic damage in an indirect way. On account of that they are negative in the main Salmonella/ microsome reverse mutation assay using TA 98 and TA 100 strains. Marginal mutagenicity was detected only in a special TA102 strain. In the conventional 6-thioguanine resistance assay on Chinese hamster V79 cells Hoechst 33342 harbors mutagenic activity at highly toxic concentrations only. In our study all bisbenzimidazols produced significant increase of wtsP4 clone frequency at non-toxic concentrations. These dyes exhibited dose-dependent blastomogenic effect, and Hoechst 33342 showed twice higher clone frequency than others that is associated with different cell-membrane penetration. We demonstrate TOPO I inhibition for all MGBLs analyzed. Surprisingly, new derivatives were mutagenic in Ames assay. Thus, our investigation of MGBL mutagenic properties demonstrates the necessity to include the tests for both point mutagenesis and recombinogenic activity determination. Drosophila SMART assay using wtsP4/+ heterozygous larvae seems to be promising in this way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2011-2731