Effects Of Nanomaterials On Chromatin Structures And Histone Modifications Within Cells

Nanotechnology, a forefront of emerging technology, is deepening and changing our understanding of the objective world, and will trigger a new industrial revolution, which thus have an important influence on the development of economy and society in the future. Nanotechnology has been applicated in various fields, In the field of medical and health care, nanotechnology will have a profound influence on early diagnosis and treatment of diseases, especially for serious illness. The ways which human exposed to nanomaterials include inhalation exposure, skin contact, swallowing contact and eye contact. The surface of a body and the internal organs is covered by epithelial cells, and the epithelial cells are firstly contacted by nanomaterials cell when the human body exposed to nanomaterials. Under various stress, the cellular stress feedback mechanism is triggered, and the chromatin structure remodeling plays an important role in response to stress. The dynamic changes between euchromatin and heterochromatin result in activating or silencing stress relevant genes which perform the cell pathway in survival and death in order to preserve the health of the individual life. All these dynamic changes are involved in the reversible regulation of histone modifications that occur quickly and flexibly without changing of DNA sequence.Considerable researches about nano biological effects and security have been done. But more studies are required in order to elucidate the complicated mechanism, especially the research about nanomaterial stress response involved in chromatin structure remodeling, which will be the important means of nano toxicity assessment. In this paper, we used normal epithelial cells (HaCaT) and cancer epithelial cells (A549) as materials and investigated the molecular genetic and histone modifications mechanism of nanomaterial induced different nucleolus disorders in normal/cancer epithelial cells. We also investigated the molecular genetic and histone modification mechanism of cell cycle arrest and apoptosis in normal epithelial cell. The results are as follows:1. Nanomaterials induce different nucleolus disorders depending on epithelial cancerizationWe studied the effect of nanomaterial composition, size and dissolution of toxic ions on cell viability. Nanomaterials included carbon nanotubes (SWCNT, MWCNT < 8nm and MWCNT> 50 nm) and metal oxides nanomaterials (TIO2 NPs and ZnO NPs),tested cells included human normal (HaCaT)and cancer (A549) epithelial cell. Cell viability assays showed cells were very susceptible to ZnO NPs, especially HaCaT cells, while cells were more resistance to TIO2 NPs. After exposed to 1000 μg/mL ZnO NPs, the viability of HaCaT cells reduced to 22.7% and A549 cells to 38.8% compared with untreated cells, while when exposed to 100 μg/mL ZnO NPs, the viability of HaCaT cells reduced to 25.6% and almost no effects on A549 cells compared with untreated cells. MWCNT< 8 nm were substantially more toxic than similar materials with bigger diameters (MWCNT> 50 nm).1000μg/mL MWCNT < 8 nm induced 16.2%reduced HaCaT cell viability and 30.0%A549 cell viability compared with untreated cells, while 1000 μg/mL MWCNT> 50nm only induced 10% reduced HaCaT cell viability and little influence on A549 cells. SWCNT had little effects on tested cells in 24h treatment. Among the five nanomaterials, materials which releasing toxic ions and providing small size exhibited the higher cytotoxicity to tested cells, the cell toxicity is material property, concentration and cell type dependent. Cellular responses were influenced by protein adsorption and material-protein interactions in nanomaterial exposure, therefore we measure nanomaterials uptake in normal and cancer epithelial cells with flowcytometry. We found different cell uptake depended on nanomaterial property, exposure time and cell type, especially metal oxides nanomaterials (TiO2 NPs and ZnO NPs) induced obviously increased uptake. Global histone methylation and demethylation were involved in cell responses to environmental stresses, and H3K9me2 was heterochromatin marker in the nucleus. We therefore analyzed dynamic changes between heterochromatin and euchromatin following various nanomaterials treatment with immunostaining. The results showed that the increased H3K9me2 signals had occurred after 3h nanomaterial exposure, which the nanomaterial uptake or surface adsorption in cells were not happened obviously compared with 24h SSC (figure 2), suggesting the response of euchromatin transforming into heterochromatin happened in the early exposed time. Then we quantified the changes between two chromatin forms with the H3K9me2 and H3K9ac signals with western bolt. Heterochromatin prevents homologous recombination between rDNA repeats to preserve nucleolar structure and rDNA stability, the RNA Pol I transcription and/or rRNA processing in nucleolus were regulated of to preserve cellular energy homeostasis.Real-time PCR results showed nanomaterial induced decreased expression of 45S rRNA precursor in normal epithelial cells and increased expression in cancer epithelial cells. In HaCaT cells, the expression of 45S rRNA precursor reduced to 1.60%,50%,18%,10%,49%after exposed to SWCNT, MWCNT< 8 nm, MWCNT> 50 nm, TIO2 NPs and ZnO NPs respectively. In A549 cells, the expression of 45S rRNA precursor increased 194.8,227.9,241.8 and 18.3 folds after exposed to SWCNT, MWCNT< 8 nm, MWCNT> 50 nm and ZnO NPs respectively. These results suggested that the different transcriptional molecular mechanism of 45S rRNA precursor in normal and cancer epithelial cells under nanomaterial stess.We analyzed histone modifications of H3K9ac, H4K5ac, H3K9me2, H3K27me2 and H3K4me2 in 45 S rDNA region with ChIP assay. The results showed the different expression of 45S rRNA precursor in normal and cancer epithelial cells were mainly asscioated with the H3K9ac, H4K5ac and H3K9me2 modifications after exposed to nanomaterials. H3K4me2 and H3K27me2 modifications may also involve in the 45 S rDNA transcriptional regulation in nanomaterial treatment, but influenced by cross-talk among various histone modifications2. Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytesWe systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis.