Mechanism Study Of Iron Oxide Nanoparticles On Intracellular Iron Metabolism And Ferroptosis Activation

Objective:Iron and steel industry and traffic exhaust emissions have become the main sources of atmospheric iron pollution.Iron particles caused cell damage by oxidative stress.Ferroptosis is a form of novel cell death caused by the accumulation of lipid peroxidation with cell membrane structural alteration.In order to explore mechanism study of iron oxide nanoparticles on intracellular iron metabolism and its relationship with activation of ferroptosis pathway,and provide a basis for comprehensive toxicity evaluation of iron oxide nanoparticles.Methods:Iron oxide nanoparticles were synthesized by co-precipitation method in hyaluronic acid hot solution,and the hydrated particle size and Zeta potential were measured.X-ray absorption near edge structure（XANES）was used to confirm the changes of intracellular iron morphologies in nanoparticles during iron metabolism.The expression of genes related to iron metabolism and glutathione metabolism（TFR,FTH,SLC7A11,SLC3A2 and ACSL4）was measured by qPCR and western blot.After exposed to iron oxide nanoparticles cells for 24 h,the intracellular lipid peroxidation was quantified by high-content fluorescence,and the morphological of mitochondria was captured by electron microscopy.Then,the cells were pretreated with ferroptosis inhibitors（Fer-1,Lip-1,DFO）,the changes of cell death level were determined by flow cytometry.Results:Iron oxide nanoparticles were spherical structures with the size of about 10nm according to the TEM characterization.The hydrodynamic size was 36.24 nm,while the zeta-potential of nanoparticles was-24.1 mV according to the potential analyzer measurement.The chemical forms of Fe during 72 h cellular uptake of nanoparticles could be determined by Fe K-edge XANES spectra.The amount of Fe²⁺increasing to 22.1%after 4 h exposure.This observation was coupled with the amount of intracellular iron retained as Fe–OOC–species increased from 74.7%to 95.2%after72 h exposure.There was a time-dependent relationship between intracellular ferritin gene expression and 24 h after exposure to iron oxide nanoparticles.Fer-1,Lip-1 and DFO could inhibit cell death,which demonstrated that the nanoparticles-induced cell death was attributed to ferroptosis.By combining proteome and transcriptome analysis,TIMM8B protein was the target of co-regulation in ferroptosis and apoptosis.During exposure to nanoparticles for 24 h,the consumption of glutathione increased and the activity of glutathione peroxidase 4 decreased,and the accumulation of intracellular reactive oxygen species was increased as confirmed by the fluorescence probe CM-H2DCFDA.The high-density mitochondrial membrane and reduction of crista structure were observed in nanoparticles treated cells,and the lipid peroxidation fluorescence signals measured by the BODIPY C11 581/591 probe gradually increased.Conclusion:Iron metabolism disorder was leaded by exposed to iron oxide nanoparticles,resulting in glutathione consumption and accumulation of lipid peroxides,eventually leading to ferroptosis.This study provides a research basis for toxicity evaluation of iron oxide nanoparticles,and provides a research basis for damage caused by exposure of iron oxide particles in the workplace.