Abstract A09: Characterization of both topoisomerase II-dependent and -independent induction of DNA double-strand breaks, damage signaling pathways, and chromosomal translocations by subgroups of bioflavonoids

Background: Etoposide is a well-characterized poison of topoisomerase II that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocation. Bioflavonoids have a similar chemical structure to etoposide, and some evidence from our lab and others suggests that they also induce DSBs and promote genome rearrangements. Since bioflavonoids are found in natural foods as well as in dietary supplements and energy drinks, quantification of their potency has important physiologic impact. The purpose of this study is to determine if subgroups of bioflavonoids induce DSBs by direct or indirect inhibition of topoisomerase II (topo II) and if the downstream damage signaling pathways and illegitimate repair are distinct. Methods: The experiments exposed a mouse embryonic stem cell Reporter cell line to physiologically relevant low and increasing doses of a panel of bioflavonoids. Following exposure, the kinetics of appearance of DNA damage was measured by immunocytochemistry and confocal microscopy to quantify γ-H2AX foci over time. Induction of DNA damage pathways was measured by Western blotting. Finally, appearance of chromosomal translocations was measured by appearance of GFP+ cells. To determine if bioflavonoid-induced DNA damage is directly through inhibition of topo II or an alternative mechanism, all experiments were performed in parallel in the presence of dexrazoxane, a topo II inhibitor. Results: Exposure to subgroups of bioflavonoids generated distinct amount of γ-H2AX foci. In addition, the kinetics of the appearance and repair of DNA damage differed between subgroups. Most bioflavonoid treatments showed persistent damage 8 hours post-exposure, though no treatment group caused as extensive damage as etoposide. Comparison of γ-H2AX foci immediately after bioflavonoid exposure with and without pretreatment with dexrazoxane demonstrated that bioflavonoids differ in their action in a topo II-dependent or -independent manner. Exposure to luteolin and myricetin after pretreatment with dexrazoxane produced a reduction in the presence of γ-H2AX foci. By contrast, exposure to genistein after pretreatment with dexrazoxane produced no change, and exposure to kaempferol or quercetin after pretreatment produced increased presence of γ-H2AX foci. Downstream induction of signaling pathways and formation of chromosomal translocations following exposure to bioflavonoids has been characterized. Current approaches are using pretreatment with dexrazoxane to also determine if signaling and repair patterns are distinct between the bioflavonoid subgroups. Conclusions: Bioflavonoids are known to induce pleiotropic effects on cells. These data suggest that the subgroup of bioflavonoid has a distinct mechanism of action to induce DNA damage in either a topo II-dependent or -independent manner, which may have implications for the potential of individual bioflavonoids to promote genome instability and chromosomal translocations. Citation Format: Donna A. Goodenow, Anindita Ghosh, Kiran Lalwani, Christine Richardson. Characterization of both topoisomerase II-dependent and -independent induction of DNA double-strand breaks, damage signaling pathways, and chromosomal translocations by subgroups of bioflavonoids [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A09.