Possible Toxicological Mechanism In Benzoyl Peroxide Induced-NDA Damage In HepG2Cells

Objective:Benzoyl peroxide (BPO) is a kind of bleaching agent which isgenerally added to flour in our country since1980s.It is used of whitening flour,accelerating the ripening of flour, slowing the speed of flour mildew to change,improving flour quality, and so on. In recent years, as a food additive, peopleincreasingly pay more attention to its potential safety problem, making its applicationcontroversial. Our country has banned using benzoyl peroxide as a food additive sinceMay2011. And around the world, there are still many countries continue to usebenzoyl peroxide as a food additive, and adding quantity varies.Current studies show that external use of BPO as a treatment for acne maybe hasthe carcinogenic potential, but the intake of BPO whether affect human health, stillnot determined. Some reports says that BPO can decompose into benzene and freeradicals when heated, so we are inspired to explore whether BPO can do harm to HepG2cell DNA by producing reactive oxygen species, and then do further study on itsmechanism. HepG2cells are derived from human liver cancer cell line, it retains manyfeatures of human normal liver parenchyma cells,and it has complete bioconversion ofmetabolic enzyme activities, it has been shown to be an ideal cell lines to detect thegenetic toxicity of exotic compound.We choose the HepG2cells line to investigate BPO-induced DNA damage and theroles of oxidative stress and lysosomal membrane permeabilization in this damage inHepG2cells, providing some information for safety assessment to human on BPO.Methods: HepG2cells were selected as test system. The single cell gelelectrophoresis assay (SCGE) was used to detect the DNA damage induced by BPO.Western blot was used to detect the expression of damage proteins.To elucidate thepossible mechanism of DNA damage caused by BPO in HepG2cells, we used2,7-dichlorofluorescein diacetate (DCFH-DA) to monitor the levels of reactive oxygen species (ROS); we chose 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 HepG2cells were pretreated with NAC, NH4Cl and desipraminefor1h. The data was analyzed by SPSS11.5.Results: BPO (100μM), for1h, caused a significant increase of the DNA damagein the SCGE. The level of intracellular ROS was significantly increased.The expressionof P53was also significantly increased with BPO50μM–100μM) for24hours.Lysosomal membrane destabilization observed in HepG2cells was a statisticallysignificant change; mitochondrial membrane potential also change after treatment withBPO (50μM–100μM) for1h. Cells pretreated NAC, NH4Cl and desipramine,theexpression of P53reduced and its DNA fragmentation induced by BPO weredecreased,meanwhile the change of intracellular ROS, lysosomal stability, whichindicated that they protect HepG2cells DNA from fragmentation.Conclusion: The results suggest that BPO caused DNA strand breaks and theexpression of damage proteins increased, which indicates that BPO did induce DNAdamage effects in HepG2cells.The toxicological mechanism probably through theincrease of ROS which cause oxidative DNA damage. After HepG2cells pretreatedwith NAC, DNA strand breaks were significantly reduced, the expression of P53reduced,suggest that DNA damage induced by BPO involved oxidative stress.Meanwhile, lysosomal membrane stability was protected when cells were pretreatedwith NH4Cl and desipramine, DNA strand breaks induced and the expression of P53reduces, which tell us that lysosomal membrane permeabilization may be themechanism of DNA damage induced by BPO and lysosomal proteases play a veryimportant part in this mechanism.