Possible Involvement Of Oxidative Stress And Lysosomal Membrane Permeabilization In Ellagic Acid Induced-DNA Damage In Human HEK-293Cells

Objective: Ellagic acid (EA) is a dietary polyphenol compound found in a widevariety of plants, including berries, fruits, nuts and vegetables. Several studies suggestedthat EA have anticarcinogenic, antioxidant and anti-inflammatory, etc. Several studieshave indicated that EA could contribute to the induction of toxic effects in cells. Theobjective of this study was to investigate EA-induced DNA damage and the roles ofoxidative stress and lysosomal membrane permeabilization in this damage in HEK-293cells, providing some information for safety assessment to human on EA.Methods: HEK-293cells were selected as test system. The single cell gelelectrophoresis assay (SCGE) was used to detect the DNA damage induced by EA. Toelucidate the possible mechanism of DNA damage caused by EA in HEK-293cells, weused2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) tomonitor the levels of reactive oxygen species (ROS) and glutathione (GSH);8-hydroxyderoxyguanosine (8-OHdG) was also measured by immunocytochemistrystaining analysis; we used Acridine orange (AO) and Rhodamine123to measure thechanges of lysosomal membrane stability and mitochondrial membrane potential. Theeffects of oxidative stress and lysosomal membrane permeabilization on DNA damagewere observed when HEK-293cells were pretreated with NAC, NH4Cl, pepstain A anddesipramine for1h. The date was analyzed by SPSS11.5.Results: EA (120μM), for1h, caused a significant increase of the DNA migrationin the SCGE. The level of intracellular ROS was significantly increased andintracellular GSH was decreased. EA at dose (60μM-120μM) caused a significantoxidative damage through8-OHdG formation in HEK-293cells for3h. Lysosomalmembrane destabilization observed in HEK-293cells was a statistically significantchange; mitochondrial membrane potential did not change after treatment with EA (30 μM–120μM) for1h. Cells pretreated NAC, NH4Cl, pepstain A and desipramine, itsDNA fragmentation induced by EA were decreased as the same as the change ofintracellular ROS, lysosomal stability, which indicated that they protect HEK-293cellsDNA from fragmentation.Conclusion: The results suggest that EA caused DNA strand breaks, whichindicates that EA induced DNA damage effects in HEK-293cells. EA exerts DNAdamage effects probably through the formation of ROS, the depletion of GSH and theincrease of8-OhdG which cause oxidative DNA damage. After HEK-293cellspretreated with NAC, DNA strand breaks were significantly reduced, we suggest thatDNA damage induced by EA involved oxidative stress. Meanwhile, lysosomalmembrane stability was protected when cells were pretreated with NH4Cl, DNA strandbreaks induced by EA were reduced by pepstain A and desipramine, which tell us thatlysosomal membrane permeabilization may be the mechanism of DNA damage inducedby EA and lysosomal proteases play an important role in this mechanism.