| Background and objective: Recently,the worsening of Fine particulate matter(PM2.5)pollution is one of the most urgent issue facing our country,and the potential health risks have attracted ever-growing the public attention.Previous epidemiologic studies have demonstrated that long-term exposure to PM2.5 has been reported to be closely related with the increased lung cancer risk and mortality in populations.In 2015,up to 23.9% of total lung cancer deaths attributed to PM2.5 pollution in China.And in 2013,the International Agency for Research on Cancer(IARC)concluded PM2.5 as a Group 1 human carcinogen,but the mechanisms underlying PM2.5-associated carcinogenesis are not well illuminated.The exploration of PM2.5 carcinogenic mechanism will provide an important basis for the development of effective intervention,risk assessment and management of PM2.5 pollution.Recent studies have revealed an important contribution of epigenetic regulation such as DNA methylation in lung cancer.Epidemiological studies have reported that PM2.5 exposure may have pleiotropic effects on DNA methylation patterns,including both gene-specific hypermethylation and global DNA hypomethylation.Therefore,it can be inferred that DNA methylation aberration could be an important part during the development of PM2.5-indcued lung cancer,which is urgently needed to be supplemented and confirmed by mechanism researches.Due to ethical limitations,existing studies have to explore the mechanisms underlying PM2.5-induced lung cancer using animal experiments or cell experiments.De facto,most in vitro experiments lunched previously prone to focus on PM2.5-induced adverse effects under frequently-used exposure conditions(short term exposure to high concentrations)and simply considered the toxicity effect of PM2.5 was the accumulation of "dose".But the toxicity mechanisms obtained from those experiments are sometimes conflictive in interpreting the course of some PM2.5-induced diseases.Proper exposure scenario based on the real-world condition could contribute to comprehensively reveal PM2.5-induced DNA methylation alteration and the role of DNA methylation aberrations in PM2.5-induced lung cancer.Thus,here we first constructed two exposure models based on two common exposure conditions in real life using human bronchial epithelial(BEAS-2B)cells to detect PM2.5-induced toxicity events and cellular responses,which aimed to present systematic comparison of toxic effects in the two exposure models and explore the possible toxicity mechanisms involved.Then we chose repeated exposure model to detect the effects of PM2.5 on global genome DNA methylation and the methylation status of P53 promoter,explore the regulation mechanisms of PM2.5-indcued DNA methylation pattern aberration and reveal the role of DNA methylation aberrations in PM2.5-induced lung cancer.The prospective findings of our study would the epigenetic mechanism underlying PM2.5-induced lung cancer,offer new insight into carcinogenic risk assessment of PM2.5,help to revise Air Quality Standards and identify specific interventions to reduce or prevent adverse outcomes induced by PM2.5.Methods: First,particle size distribution,the stability analysis of PM2.5 in culture media and morphological characterization of PM2.5 were determined by NANOPHOX instrument,Turbiscan Lab? Expert and transmission electron microscope(TEM),respectively.Then we designed two exposure models,24-h exposure to high-doses PM2.5(0,6,12,24,48,96 μg/cm2)and 10 days’ repeated exposure to low levels of PM2.5(0,1.5,3,6 μg/cm2)using MPPD software.After treatment with PM2.5,the cytotoxicity of PM2.5 was assessed by CCK-8 assay and LDH release.The ultrastructure change of cell injury were also observed TEM.CM-H2 DCFDA probe was also employed to detect intracellular ROS production.Oxidative damages of DNA,cell cycle distribution and cell death in Beas-2B cells were evaluated by comet assay,flow cytometry using Annexin-V/PI double staining or PI staining,respectively.Western blot was performed to detect the expression of the proteins involved in DNA damage repair,autophagy,mitochondria function and inflammation,epigenetic regulation.Cellular ATP content was measured by a luciferase-based assay.ER-Stress,UPR and the mRNA expression of P53 were determined by qRT-PCR.DNA methylation levels of global genome and P53 promoter were detected using colorimetric assay and BSP,respectively.And EMSA was used to analyze the effect of methylated cytosine on the binding between DNA probes and nuclear proteins.Additionally,siRNA silencing,pretreatment with NAC or specific-inhibitors were performed to epxplore the mechanism for PM2.5-indcued cell necrosis in the acute exposure model and detect the role of ROS-Akt-DNMT3 B signal pathway in the repeated exposure model.Results: 1.The average diameter of PM2.5 was about 0.74 ± 0.08 μm.The particles displayed an approximately normal size distribution and good stability in DMEM medium.TEM images revealed the multi-shape and multi-size morphological characterization of PM2.5.2.Under acute exposure model,high concentrations of PM2.5 caused ROS burst,LDH release and marked DNA damage.TEM results also showed that PM2.5 exposure indcued obvious mitochondria swelling,vacuolization,and dilation of endoplasmic reticulum.The protein level of PARP-1(116 kDa)and P21,and the ratio of phospho-P53/P53 and γH2A.X/H2 A.X in cells were all gradually increased.But flow cytometry results did not detect any change in cell cycle arrest and apoptosis,as well as cleaved Caspase-3.The percentage of necrotic cells and IL-6 levels were increased in a dose-dependent manner.Western bolt and TEM showed PM2.5 could induce autophgy through mTOR-Beclin1-LC3 B signmal pathway and increase P62/SQSTM1 expression.In addition,PM2.5 caused dysfunction of ER-stress and UPR,which were confirmed by anaylysis of GRP78 and three sensor genes expression.PM2.5 also increased the protein level of NRF-1,mtTFA and PGC-1α,but cellular ATP contents were dramatically decreased.Besides,acute PM2.5 exposure increased DNMT1 and SIRT1 levels but decreased DNMT3 B level both in a dose-dependent manner.3.Exposure to high-dose of PM2.5 increased the expression and mitochondrial transposition of HO-1,which consequently reduced the release of cytochrome C from mitochondria to cytosol.Pretreatment with 3-MA or HO-1 siRNA could independently reduce PM2.5-triggered cell necrosis and promoter apoptotic cell death.4.In contrast,under repeated exposure model,low levels of PM2.5 led a sustained,low-grade elevation of ROS in cells,following a mild form of mitochandria swelling in all exposure groups and DNA damage in the 6μg/cm2 group.Repeated exposure to PM2.5 enhanced the level of the ratio of γH2A.X to H2 A.X,cleaved PARP-1(89 kDa),but the expression levels of phospho-P53,P53 and P21 were actually decreased in a dose-dependent manner.PM2.5 also induced cell cycle arrest at S phase,increased cleaved Caspase-3 protein expression and promoted cell apoptosis and cellular IL-6 content.TEM observed AVds,lamellar bodies but Western Blot did not detect any perturbation of the mTOR-Beclin1-LC3 B pathway in treated cells.Furthermore,PM2.5 activated ER-Stress and UPR through GRP78 and its sensors,but inhibited NRF-1 expression and slightly lowered cellular ATP levels.In addition,chronic PM2.5 treatment gradually inhibited DNMT1 protein expression but enhanced DNMT3 B protein level in cells.5.Repeated exposure to low levels of PM2.5 led to sustained low-grade ROS accumulation.After 5-days exposure,low levels of PM2.5 significantly decreased DNMT1 protein expression and global genome DNA mtheylation levels,while increased cellular DNMT3 B and phospho-Akt levels in a dose-dependent manner.But we did not detect obvious PM2.5-induced DNA damage,cell cycle arrest and apoptosis.Repeated exposure to low-levels of PM2.5 for 10 days,we found the increase in methylated cytosine bases in P53 promoter and the reducation in P53 expression in cells.Furthermore,pretreatment with NAC or LY294002 could decrease Akt phosphyrationand DNMT3 B expression,inhibit P53 promoter hypermethylation,and enventually restore P53 protein expression in BEAS-2B cells.Conclusion: 1.These results indicate that PM2.5 induced distinct forms of cellular responses and toxicity in the two exposure scenarios.Acute exposure to high concentraions of PM2.5,extreme intercellular oxidative damage beyond the elicited stress responses would result in the unbalance of homeostasis and an unanticipated collapse effect,induce acute respiratory inflammatory symptoms.In contrast,repeated exposure to low doses of PM2.5 displays no obvious cytotoxicity,but persistent low-grade of oxidatvie stress and inflammatory produce a series of abnormal adaptive responses,which enventually cause related diseases.All of these suggest that “exposure scenario” maybe another important factor determining the toxic effects of PM2.5.2.Acute exposure to high concentrations of PM2.5 causes autophagy-related cell necrosis.The decrease in cytochrome C release and apoptosis by upregulation of HO-1 maybe assist PM2.5-induced autophagy-related cell necrosis.The results reveal dual roles for HO-1 in PM2.5-induced cytotoxicity and present a possible explanation for the onset of acute respiratory symptoms under extreme particulate air pollution.3.Sustained low-level increase in ROS induced by repeated PM2.5 exposure could induce global genome DNA hypomethylation through down-regulating DNMT1 protein expression,cause P53 promoter hypermethylation through ROS-Akt-DNMT3 B signal pathway,decrease P53 expression in BEAS-2B cell.All of these events enventually trigger and promote the development of lung cancer. |
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