Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms

Abstract

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Keywords: DNA damage; Double-strand break; Environmental mutagenesis; Etoposide; Genome instability; Topoisomerase II.

Figure 1.. Bioflavonoids Stimulate DNA DSBs and Kinetics of DNA Damage and Repair.

Cells were treated with A, etoposide (Eto), B, genistein (G), C, quercetin (Q), D, kaempferol (K), E, myricetin (M), or F, luteolin (L) at a range of doses before being stained for γH2AX foci either immediately after treatment or after 1, 4, or 8 h post-exposure. Doses for etoposide include 6.25, 12.5, and 18.75 μM. Doses for genistein, quercetin, kaempferol, and myricetin include 25, 50, 75, and 100 μM. Doses for luteolin doses include 50, 100, 150, and 200 μM. Each point represents the number of foci in one cell, a minimum of 100 cells were examined in each group. The mean is displayed with the 95% confidence interval as error bars. All p-values are provided in Supplemental Table 1.

Figure 2.. Combinations of Bioflavonoid Exposures Display Similar DNA DSBs and Kinetics to Singular Bioflavonoid Exposures.

Cells were treated with A, genistein/quercetin (G/Q), or B, genistein/quercetin/luteolin (G/Q/L) at a range of doses before being stained for γH2AX foci either immediately after treatment or after 1, 4, or 8 h post-exposure. Doses for genistein/quercetin include 25/25, 50/50, 75/75, and 100/100 μM and for genistein/quercetin/luteolin doses include 25/25/50, 50/50/100, 75/75/150, and 100/100/100/200 μM. Each point represents the number of foci in one cell, a minimum of 100 cells were examined in each group. The mean is displayed with the 95% confidence interval as error bars. All p-values are provided in Supplemental Table 1.

Figure 3.. Dexrazoxane Pre-treatment Reduces Bioflavonoid-induced DNA DSBs Through Top2-dependent Mechanisms.

Cells were pre-treated with dexrazoxane at 200 μM for either 1 or 5 h before 1 h treatment with A, etoposide (Eto) 6.25 μM, B, myricetin (M) 50 μM, C, quercetin (Q) 75 μM, D, genistein (G) 75 μM, E, luteolin (L) 100 μM, F, kaempferol (K) 100 μM, G, genistein/quercetin (G/Q) 75/75 μM, or H, genistein/quercetin/luteolin (G/Q/L) 50/50/100 μM. Immediately after treatment, cells were stained for γH2AX foci. Each point represents the number of foci in one cell, a minimum of 100 cells were examined in each group. The mean is displayed with the 95% confidence interval as error bars. * p<0.05, *** p<0.0001