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) in Salmonella typhimurium (S. typhimurium) TA1535. With the aim to clarify the antimutagenic mechanism of these flavonoids, we evaluated the noncovalent interactions of test compounds (LCD, ILTG, ethidium bromide (EtBr), or Hoechst 33258) with calf thymus DNA (ctDNA) using a UV-Vis spectrophotometer and quantified the DNA adducts formed when treated with a mixture of MNU and the test compounds. Additionally, we measured the half-life of MNU and the amount of each test compound remaining in the reaction mixture.

RESULTS: The spectral and thermodynamic parameters indicated considerable binding between flavonoids (LCD and ILTG) and DNA. Intercalative EtBr and the minor-groove binder Hoechst 33258 inhibited MNU-induced mutagenicity in S. typhimurium TA1535. O6-Methylguanine (O6-MeG) and N3-methyladenine (N3-MeA) were quantified in a mixture of ctDNA and the test compounds (LCD, ILTG, EtBr, and Hoechst 33258) using LC‒MS/MS. The amount 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 flavonoids remaining in the reaction mixture were observed, indicating that LCD and ILTG did not react directly with MNU.

CONCLUSIONS: LCD and ILTG directly bind to DNA and suppress DNA adduct formation. However, interaction with DNA alone does not fully account for their antimutagenic activity, implying the involvement of other mechanisms.