The Effects Of 1,2,5,6,9,10-Hexabromocyclododecanes (HBCDs) On RXR?PXR?PPAR? And Its Neurotoxic Effects Preliminary Discussion

Backgrouds: Hexabromocyclododecanes,as a new type of persistent organic pollutants,are widely present in the atmosphere,soil,sediments,water and various organic matters.It has been widely used in polystyrene foam,interior decoration textile and electronic products because of its low consumption,good flame retardant effect,little influence on the physical properties of materials and good thermal stability.The main toxic target organ of HBCDs is the neuroendocrine system and the reproductive development system.The commercial HBCDs are composed of three diastereoisomer s: ?-,?-,?-HBCD.At present,there are few studies on the toxicity of the three isomers of HBCDs,especially the toxicity of three isomers to neuron cells has not been reported.Objectives: To investigate the effects of HBCDs on cell proliferation and oxidative damage of mouse neuroblastoma N2 a cells by in vitro experiments.Furthermore,to explore the possible molecular mechanisms from the aspects of expression,localiza tion and interaction of three important cellular nuclear receptors: retinoid X receptor(RXR?),peroxisome proliferator activated receptor(PPAR?),and pregnane X receptor(PXR).Thus,this thesis work reveals the molecular mechanism of HBCDs neurotoxicity and provides scientific evidence for other toxic effects of HBCDs.Methods: N2 a cells were treated with different concentrations of three diastereoi somers of HBCDs: ?-hexabromocyclododecane(?-HBCD),?-hexabromocyclododecan e(?-HBCD),and ?-hexabromocyclododecane(?-HBCD).The cytotoxicity of three dia stereoisomers of HBCDs on N2 a cells was detected by CCK-8.The effects of three HBCD isomers on the cell cycle of N2 a cells were analyzed by flow cytometry.The mRNA and protein level expression of mouse 8-oxoguanine DNA glycosylase(m O GG1)were determined by real-time polymerase chain reaction(RT-PCR)and western blotting(WB).The activity of lactate dehydrogenase(LDH)in N2 a cells was deter mined by micro-enzyme method.The content of malondialdehyde(MDA)was determined by TBA method.The glutathione peroxidase(GSH-PX)activity was determined by colorimetry.Intracellular ROS levels were measured by DCFH-DA(2,7-dichlorofu orescin diacetate).The mRNA and protein level expression of RXR?,PPAR?,PXR and its downstream target gene cytochrome P450 subunit CYP3A11,were detected by RT-PCR and western blot.The interactions between RXR?,PXR and PPAR? receptors were analyzed by immunoprecipitation techniques.Results: ?-HBCD and ?-HBCD have obvious cytotoxic effects on mouse neurob lastoma N2 a cells.The cytotoxicity of the three HBCD isomers on N2 a cells was ?-HBCD > ?-HBCD > ?-HBCD.Twenty-four hours are the most appropriate time for HBCDs exposure.The IC50 of ?-HBCD,?-HBCD to N2 a cells were 60.07 and 10.52 ?mol/L,respectively.However,the cytotoxicity of ?-HBCD for the N2 a cells were not determined in this work.?-HBCD and ?-HBCD induced oxidative damage in N2 a cells by increasing LDH leakage rate,MDA content,mOGG1 level,ROS level,and decreasing GSH content.?-HBCD and ?-HBCD blocked the cell cycle at G2/M phase.The expression of RXR?,PPAR?,PXR,CYP3A11 at mRNA and protein levels were significantly increased after the cells exposure to ?-HBCD and ?-HBCD for 24 hours(P<0.05).The results of immunoprecipitation showed that there exists interactions between RXR?,PPAR? and PXR in N2 a cells before and after ?-HBC D and ?-HBCD exposure.Conclusions: ?-HBCD and ?-HBCD have significant cytotoxicity effects on N2 a cells,leading to oxidative damage of N2 a cells.?-HBCD and ?-HBCD can interfere with normal cell cycle and blocked cell cycle at G2/M phase,which inhibit cell proli feration.?-HBCD,?-HBCD can up-regulate the expression levels of RXR?,PPAR?,and PXR.Meanwhile,the expression of CYP3A11,which was downstream target gene of PXR was also significantly increased.There were always interactions between RXR?,PPAR?,and PXR in N2 a cells before and after ?-HBCD and ?-HBCD exposure.These results suggest that RXR?,PPAR? and PXR may mediate the neurotoxiceffects of HBCDs,and the molecular mechanism of receptor interaction needs to be further explored in the future.