Analysis The Mechanisms Of Parthanatos-like Cell Death,Ferrinophagy And Ferroptosis Induced By Polybrominated Biphenyl Hydrazine

2-(2’,4’-Bromophenoxyl)-benzoquinone(PBDEQ)is the metabolite of the halogenated aromatic hydrocarbon compound polybrominated diphenyl ether(PBDES),which are widely used in the biological system and abiotic system as brominated flame retardants.It has been shown to be a severe health hazard to human.PBDEQ have higher biological activity than aromatic hydrocarbons due to the special structural characteristics of quinones.In general,quinones(e.g.1,4-benzoquinone,polychlorinated biphenyl hydrazine,bisphenol A hydrazine,polycyclic aromatic hydrocarbon hydroquinone)are able to achieve a Michael addition reaction with DNA.In addition,quinones can also induce oxidative stress response by semi-cluster free radicals or downstream reactive oxygen species produced by hydroquinonesemiquinone-quinones in the redox cycling,which leading to cytotoxicity,genotoxicity,and other effects ultimately.Part Ⅰ: PBDEQ induces genotoxicity in Hela cells.In this chapter,the experimental evidence shows that PBDEQ causes DNA oxidative damage by producing a large unmber of reactive oxygen species.Firstly,we synthesized 2-(2’,4’-bromophenyl)-benzoquinone(PBDEQ)according to the method described by Huang Lihua.Then,we used the single cell gel electrophoresis(SCGE)assay to detect DNA damage,and the results showed that the PBDEQ group had a significant comet tailing and serious DNA damage had been elicited.The micronucleus assay was used to detect chromosome damage.The experimental results showed that the micronucleus rate of PBDEQ group was significantly increased,indicating that PBDEQ damaged the cell chromosome severely.The 8-OHdG release assay was used to detect the degree of DNA oxidative damage.It was found that the amount of 8-OHdG in the cells was significantly increased by PBDEQ stimulation,indicating that PBDEQ could induce DNA oxidative damage in cells.Finally,we used the western blot assay and immunofluorescence assay to detect the expression of theDNA double-strand break marker γ-H2 AX.It has been found that PBDEQ activates H2 AX and increases the expression of γ-H2 AX in cells,which also indicated that PBDEQ can induce DNA double chain breaks which cause severe DNA damage.These results showed that PBDEQ can induce severe DNA damage and chromosome damage in Hela cells.Part Ⅱ: PBDEQ activates PARP-1 mediated parthanatos-like cell death via ROS.In the first part,PBDEQ had been shown to have severe DNA damage,but further study was needed to explore the toxic effects caused by irreversible DNA damage.In this chapter,the CCK8 assay and LDH release assay were performed to detect the cytotoxicity of PBDEQ in Hela cells.The results showed that PBDEQ could cause cell death and cell membrane damage.Subsequently,it was found that a large number of intracellular reactive oxygen species were produced under PBDEQ stimulation,and the mitochondrial membrane potential decreased,these two discoveries proved that PBDEQ caused severe oxidative damage to Hela cells.Besides,the flow cytometry was used to detect apoptosis induced by PBDEQ.This test indicated that the cells were expressed as Annexin V-FITC+/PI+ and positively expressed in TUNEL assay.This result showed that,except apoptosis and necrosis,PBDEQ has the ability to induce a new cell death pathway.Parthanatos cell death is a newly discovered programmed death pathway closely related to DNA damage in recent years.The western blot assay and immunofluorescence assay were used to detect the expression of marker proteins in parthanatos cell death.The results showed that PARP-1,AIF,and PAR experienced an inverted U-shape expression under PBDEQ stimulation,and the maximum expressions of PARP-1,AIF and PAR were found at the PBDEQ concentration of 5μM.Furthermore,PBDEQ promoted the transfer of AIF from the cytoplasm to the nucleus.These results indicated that PBDEQ activated the parthanatos-like cell death pathway.Finally,the experiment shows that the addition of antioxidant NAC and the presence of PARP-1 inhibitor 3AB can significantly inhibit the expression of PARP-1,AIF,PAR,and nuclear transfer of AIF.It was indicated that ROS production and activation of PARP-1 mediated PBDEQ-induced parthanatos-like cell death.However,the caspase family inhibitor Z-VAD-FMK unaffected the PBDEQ-induced elevated PARP-1 AIF,PAR,and the AIF nuclear transfer.This result suggested that the caspase family is not the key factor of the PBDEQ-induced activation of parthanatos-like cell death.In summary,PBDEQ is able to induce genotoxicity.PBDEQ-induced severe DNA damage activated the parthanatos-like cell death pathway.The occurrence of parthanatos-like cell death was regulated by oxidative stress response and excessive activation of PARP-1.It was worth noting that the parthanatos-like cell death pathway activated by PBDEQ was not regulated by the caspase family,which was different from the apoptosis.Part Ⅲ: PBDEQ induces ferritinophagyIn this study,it was discovered that PBDEQ also activated the oxidative stress response in PC12 cells,and ROS activated autophagy.Firstly,western blot assay was carried out to test the autophagy marker protein LC3-II and ATG5.The expression levels of these proteins were increased significantly at the concentration of PBDEQ of 10 μM.Moreover,NAC significantly inhibited the PBDEQ-induced expression of LC3-II and ATG5.The PBDEQ induces autophagosome-lysosome fusion was detected through immunofluorescence experiments.These experimental results demonstrated that PBDEQ is able to induce autophagy through ROS.There were studies shown that the occurrence of ferrtinophagy requires that NCOA4 participate in the bind of specifically recognize ferritin and autophagosome-lysosome.Through immunofluorescence assay,it was found that intracellular FTH,LAMP2,NCOA4,and LC3 B were co-localized by PBDEQ stimulation.This indicated that PBDEQ induces FTH binding to NCOA4 and it promotes the transfer of ferritin to lysosomes.It was found that NCOA4 was down-regulated with gradient expression by PBDEQ stimulation through western blot assay.However,FTH showed a U-shape expression by PBDEQ stimulation,and the lowest expression level was found with 10 μM PBDEQ.Moreover,PBDEQ induced an increase of the mRNA level of FTH and NCOA4.Autophagy inhibitors(CQ)could inhibit the degradation of autophagosome-lysosome.CQ could significantly increase LC3 B and FTH levels that induced by PBDEQ,and inhibited down-regulation of NCOA4 by PBDEQ-mediated,which fully demonstrated that PBDEQ induced ferritinophagy.Furthermore,inhibition of NCOA4 expression by transfection of NOCA4 siRNA inhibited the FTH reduced level by PBDEQ-induced.These results demonstrate that NCOA4 plays an important role in PBDEQ-induced ferritinophagy.Part Ⅳ: PBDEQ induces the occurrence of ferroptosis.In this chapter,the iron staining assay and the iron fluorescent probe labeling assay were performed to detect the PBDEQ-induced increase of intracellular free iron.Western blot assay result showed that PBDEQ promoted elevated expression of transferrin FPN and decreased expression of iron input protein DMT.This also proved that PBDEQ increases the intracellular free iron content.It was detected that PBDEQ induced mitochondria reduction and induced a decrease in GPX4 expression and an increase in ASCL4 expression by western blot assay.Furthermore,fer-1 significantly reduced PBDEQ-induced ferroptosis.This indicates that the PBDEQ induced the occurrence of ferroptosis.NAC inhibited PBDEQ-induced expression of ferroptosis-related proteins,indicating that PBDEQ-induced ferroptosis is dependent on the ROS production.Moreover,PBDEQ-induced ferritinophagy can promote ferroptosis.The above studies showed that PBDEQ induced ferritinophagy and ferroptosis in PC12 cells,and the occurrence of ferritinophagy promoted the occurrence of ferroptosis.