Toxicity Effects And Epigenetic Changes Induced By Treatment Of A549 Cells With Nanomaterials

Recently,nanomaterials are widely used in industry,manufacturing and medical fields,they can be absorbed by human body through inhalation and exposure,therefore the toxicity effects induced by various nanomaterials have attracted much attention and research.Here we systematically explored the toxicity effects of long-term treatment induced by three kinds of nanomaterial,the recoverability of cell viability after removal of the nanomaterial,and the changes of histone epigenetic regulation in A549 cells at the epigenetic and molecular levels.Our study focused on a series of physiological and biochemical responses of cells within 15 days of treatment and associated epigenetic modifications.Carbon nanotubes(MWCNTs and SWCNTs)and metal oxide nanoparticles(TiO2 NPs)are widely applied now.The concentration and release of carbon nanotubes in the environment are increasing,and the biological safety needs to be assessed.We treated A549 cells with MWCNT,TiO2 NPs and SWCNT at 100?g/ml.Cells exposed to various nanoparticles for 5days,10days,15days,recovery for 5days after 5days' treatment and recovery for 5days after 10days'treatment,respectively.Cell viability test results showed that the survival rates of the treated cells were generally lower than the control group but in varying degrees,indicating that the abilities of nanomaterials to inhibit cell growth were different.The results of inhalation experiments showed that different kinds of nanomaterials exerted varying extent of particle uptake and release,especially TiO2 NPs.Then we examined the levels of reactive oxygen species,the degree of lysosomal membrane destabilization,DNA damage in different treatment groups and recovery groups of each nanomaterial.The results showed that the treatment of all kinds of nanomaterials gave rise to the increase of intracellular reactive oxygen species,lysosomal membrane destabilization and DNA double-strand breaks.After removal of nanomaterials,the recoverability of cell viability varies.The cells treated with TiO2 NPs could be recovered to the same state as normal cells,and the levels of cytotoxicity and genotoxicity were decreased.However,cells exposed to MWCNT still showed a high level of toxicity effects after transferring to the normal culture medium.It was proved that MWCNT induced the most serious damage cytotoxicity and genotoxicity at the same concentration and treatment time than SWCNT and TiO2 NPs.Based on the above experimental results,we speculate that different types of nanomaterials after being absorbed into the cells disrupted the normal functions of mitochondria,lysosomes and other organelles,and to exerted pressure to the nucleus,resulting in DNA double-strand breaks and a series of toxicity effects.The treatment times and type of nanomaterials are critical determinants of the degree of cytotoxicity genetoxicity.When the nucleus is subjected to adverse external stimuli,changes in histone modification such as lysine residue modification can cause remodeling of the nuclear chromatin architecture.In order to investigate the changes of epigenetic modifications in the process of genotoxicity induced by nanomaterials,we examined the changes of the expression levels of histone H3K9me2,H3K9ac,H4K5ac and H3K4me2 in different treatment groups and recovery groups of each nanomaterial.The results indicated that four marker epigenetic modifications were in the dynamic changes compared with the control group.It was proved that the nucleus chromatin structure constantly changed between the state of euchromatin and heterochromatin due to external stimuli.Then we examined the cell cycle progression in different treatment groups and recovery groups of each nanomaterial.The results indicated that the treatment of different kinds of nanomaterials caused cell cycle arrest at different stages such as Gi phase and G2/M phase respectively.In order to further determine whether transcriptional activation and repression was correlated with dynamic changes in histone H3 modification at the promoters and transcribed regions of cell proliferation gene,chromatin immunoprecipitation using H3K9me2 antibody and real time PCR which is used to detect the expression of PCNA gene were performed.Proliferating cell nuclear antigen(PCNA)is a key gene that regulates eukaryotic DNA replication,damage repair,cell cycle progression and other biological events,and can also serve as a marker of cell proliferation.Histone H3K9me2 is a vital heterochromatic mark which can affect the process of gene transcription and cause gene silencing.Quantitative real-time PCR analysis revealed that the expressions of PCNA gene were decreased in all kinds of nanomaterials.ChIP results showed that the methylation degrees of histone H3K9me2 in PCNA gene promoters and transcribed regions treated by three kinds of nanomaterials differed from one another,suggesting that H3K9me2 histone modification may be involved in the transcriptional expression of PCNA gene.Based on the above results,we can deduce that:The nanomaterials entered the cell,disrupted the normal function of the organelles such as mitochondria,lysosomes,and exerted pressure on the nucleus,inducing DNA damage,elevating levels of reactive oxygen species,lysosomal membrane destabilization and other toxicity effects,eventually leading to cell cycle arrest.At the same time,epigenetic modification was accompanied by the process of nuclear stress response induced by nanomaterials.It was involved in the transcriptional regulation of key genes such as cell proliferation gene PCNA and dynamically regulated chromatin structure.The result of nanomaterials acting on cells eventually leads to cell cycle arrest,cell growth slows down or even death.The toxicity effects of A549 cells induced by nanomaterials depend on the type of material,the concentration of treatment and the treatment time.After removal of nanomaterials,the recoverability of cell viability was different.