Background: Ellagic Acid (EA) is a polyphenolic compound that is classified in the natural antioxidants group. Polyphenolic compounds that exert antioxidant activity possess particular importance for scientists, food producers and consumers due to their positive effects on human health. However, despite considerable evidence that EA shows antigenotoxic activity by binding to DNA, there is no systematic genotoxicity study of this substance, which can covalently bind to DNA. This study aims to reveal the possible genotoxic activity of EA using widely accepted assays for the assessment of DNA clastogenic activity: sister chromatid exchange, chromosome aberration, micronucleus and comet assays as well as to predict the interactions among EA and DNA through molecular docking.
Methods: Different assays were carried out to identify the clastogenic activity of EA on human lymphocyte DNA using Sister Chromatid Exchange (SCE), Chromosome Aberration (CA), Micronucleus (MN) and single-cell gel electrophoresis (SCGE/comet) assays. For this aim, human peripheral blood lymphocytes were treated with EA (60, 80 and 100 μg/ml) for 24 and 48 hrs in the SCE, CA and MN assays and for 1 hr in the comet assay. Furthermore, molecular docking experiments were also performed to calculate the binding energy of EA on human B-DNA structure (B-DNA dodecamer) as well as to predict noncovalent interactions among these macromolecules.
Results: At the concentrations and treatment times (24- or 48-hr) tested, EA did not induce either SCE or Chromosome Aberrations (CAs) as compared to the negative and solvent controls. Although EA slightly increased the percentage of Micronucleated Binuclear (%MNBN) cells as well as the percentage of Micronucleus (%MN) in 24 or 48-hr treatment periods at all concentrations, this increase was not statistically significant as compared to both controls. The effect of EA on DNA replication (nuclear division) was determined by the Proliferation Index (PI), the Nuclear Division Index (NDI) and the Mitotic Index (MI). No statistically significant differences were observed in the PI or NDI in 24- or 48-hr treatment periods in human lymphocyte cultures treated with EA at various concentrations. EA generally had no significant effect on the MI, as observed with the PI and NDI.
Discussion: Although the concentrations of 60 and 80 μg/mL at a 24-hr treatment period and the concentrations of 60 μg/mL and 100 μg/mL at 48-hr treatment period generally decreased the MI, those decreases were not statistically significant when compared to negative and solvent controls. Moreover, none of the concentrations of EA tested in this study were able to increase DNA damage determined by the tail DNA length, %DNA in tail and tail moment parameters in the comet assay. Although the amount of DNA damage in the comet assay decreased with increasing concentrations of EA, this decrease was not statistically significant as compared to both controls. However, molecular docking experiments interestingly showed that the binding free energy of EA with B-DNA was -7.84 kcal/mol-1, indicating a strong interaction between the two molecules.
Conclusion: Although the findings of our study show that EA does not have genotoxic potential in human chromosomes, molecular docking experiments revealed strong hydrogen bonding between EA and B-DNA molecules. Therefore, it has been proposed that the prevailing information suggesting that the molecules that bind to DNA cause genotoxic effects should be reconsidered from a wider perspective.
Keywords: Ellagic acid, sister chromatid exchange, chromosome aberration, micronucleus, single-cell gel electrophoresis, molecular docking.