Cytotoxicity Study On Fine Particulate Matter And Its Harmful Substances

Objectives:The mechanism of particulate matter(PM2.5)and its main components-induced health risks remains elucidated.In order to reveal the potential mechanism of PM2.5-induced endothelial dysfunction,we investigate the proteomic affects of PM2.5 on Human Umbilical Vein Endothelial Cells by using two-dimensional electrophoresis in conjunction with mass spectrometry.On the other hand,we further investigate the potential immunosuppression of polycyclic aromatic hydrocarbons and their derivatives on immune cells,which is enriched in PM2.5.This study aims to clarify the mechanism of fine particulate matter and its harmful substances-induced cytotoxicity from the perspective of endothelial dysfunction and immunosuppression.Metheods:In this study,Human umbilical vein endothelial cells(HUVEC)were treated with 0,50 and 100 μg/mL PM2.5 for 24 h respectively.Protein was extracted and used to investigate alterations in the protein profile using two-dimensional electrophoresis in conjunction with mass spectrometry.We screened the differentially expressed proteins,analyed the molecular biological functions,and verified these function with the vitro experiment.In the second section,we choose three compounds as the target chemicals,namely,Fluo,1-Nitro,and 9-Fluo,which were accounted of the maximum concentrations in their faimilies of PAH,NPAH and OPAH in PM2.5,respectively.The RAW264.7 cells were treated with 0,10,30,50 and 80nM Fluo,1-Nitro and 9-Fluo for 24h respectively.MTT method and extracellular LDH leakage were used to detect the proliferation vitality.The release of pro-inflammatory factors in the culture medium was detected according to the manufactory guidelines.The ROS level,MDA content and SOD activity was measured as the endpoints of oxidative damage.The three PAHs were docked into the active pocket of AhR using a ligand Fit program and fit at the binding pocket with different orientations.Real-time PCR were used to measure the transcription of AhR-mediated genes CYP1A1 and CYP1B1.A potential estimate was used to assess the correlation between inflammation,oxidative stress and cytotoxicity.In the circumstance of infections,cells were treated with the three compounds and LPS for 6h,Flow cytometer were used to measure the phagocytosis of RAW264.7.Real-time PCR were used to measure the transcription of inflammation factors.Finally,we estimated the different effects of the three PAHs on RAW264.7 cells.Results:(1)The results shown that a total of 31 proteins exhibited statistically significant changes after the treatment with PM2.5,and the main biological procession of these differentially proteins were involved in DNA damage,cell apoptosis,cell proliferation etc.Results of the vitro experiments shown that PM2.5 inhibited the SOD activity,increased the ROS level and MDA content in a concentration-dependent manner.Finally,the production of DNA oxidative damage product 8-OHdG was increased after exposure to PM2.5.The expression of DNA damage repair protein was disturbed,which promote the apoptosis of HUVECs.(2)The results shown that Low-molecular-weight PAH Fluoranthene(Fluo,PAH)and 1-Nitropyrene(1-Nitro,NPAH)induced a slight cytotoxicity at real environmental levels,while 9-Fluorenone(9-Fluo,OPAH)shown no cytotoxicity on RAW264.7 cells.Result of Molecular docking shown that Fluo formed a π-πinteraction with the Phe12 residue of AhR.It inhibited the transcription of CYP1A1 and CYP1B1 and ultimately induced the inflammatory cytokines in RAW264.7.The 1-Nitro formed both a π-π interaction and a hydrogen bond with AhR,stimulated CYP1A1 transcription,while suppressed the cytokine levels.Additionally,the inflammation potency caused by TPAHs was highly correlated with the cytotoxic potency rather than the oxidative stress potency.When stimulated by LPS,the transcription of IL-6 was inhibited by Fluo,and 1-Nitro suppressed both IL-6 and TNF-α transcription.Furthermore,only 1-Nitro gave a significant inhibition on phagocytosis.The effects of 9-Fluo on macrophages remained insignificant throughout the study since the low affinity for AhR,which resulted in low cytotoxicity.Conclusions:This study aimed to investigate the potential toxic mechanism of PM2.5 and its harmful components on HUVEC cells and RAW264.7 cells.Conclusions were listed as the follows:(1)The damage mechanism of PM2.5 on HUVECs is involoved in many pathways.① PM2.5 have a significant influence on the expression of 31 proteins which involved in DNA damage,cell apoptosis and other biological functions.②The results of the vitro experiments indicated that the toxic effect of PM2.5 on HUVEC mainly through oxidative stress pathways-induced DNA oxidative damage and its repairing system,which finaly lead to the apoptosis and HUVEC dysfunction.(2)LMW PAHs have detrimental effect on RAW264.7 cells even at real environment level.① The results suggested that LMW PAHs tended to cause mild inflammation when they bind without activating AhR.② During infection,AhR ligands caused immunosuppression and this potency for TPAHs may be higher in AhR activator than that in AhR inactivator.