Mechanistic investigation of flavonoid-mediated inhibition of N-methyl-N-nitrosourea-induced mutagenicity via noncovalent DNA binding

Background : Previous research using the Ames assay showed that licoricidin (LCD) and isoliquiritigenin (ILTG) can block mutations caused by N -methyl- N -nitrosourea (MNU)-induced mutagenicity in Salmonella typhimurium ( S. typhimurium ) TA1535. To clarify the antimutagenic mechanism of these flavonoids, we aimed to evaluate their noncovalent interactions with the test compounds (LCD, ILTG, EtBr, or Hoechst 33258) and calf thymus DNA (ct DNA) using a UV-Vis spectrophotometer and quantify the DNA adducts that formed a mixture of MNU and the test compounds. Additionally, we measured the half-life of MNU and how much of each test compound remained in the reaction mixture. Results : The spectral and thermodynamic parameters suggest considerable binding between flavonoids (LCD and ILTG) and DNA. Intercalative ethidium bromide and the minor-groove binder Hoechst 33258 inhibited MNU-induced mutagenicity in S. typhimurium TA1535. O ⁶ -Methylguanine ( O ⁶ -MeG) and N ³ -methyladenine ( N ³ -MeA) were quantified in a mixture of ct DNA and the test compounds (LCD, ILTG, EtBr, and Hoechst 33258) using LC‒MS/MS. The level of the DNA adducts decreased with increasing concentrations of the compounds. Moreover, the half-lives of MNU were similar in the presence and absence of flavonoids (LCD and ILTG). No new products were detected, and no significant changes in the remaining flavonoids were observed. These data indicate that LCD and ILTG did not react directly with MNU. Conclusions : The present study indicates that the antimutagenic effect of LCD and ILTG is likely mediated by their binding to duplex DNA, which reduces MNU-induced mutagenicity and DNA methylation.