Nm-TiO₂,nm-CuTiO₂ And Nm-Betaine Stress Effects On Gene Expression Of Physarum

According to the definition of ASTM, nanomaterials is material of which particles at least one dimension in the nanoscale?1-100 nm?. Nanomaterials has extremely small dimensions and has some special properties that others are not available, such as small size effect, surface effect, quantum size effect and quantum tunnel effect. These materials hold great interests in industrial and medical fields, but also cause biological toxic to varying degrees, including the destruction of cellular ultrastructure, cytoskeleton, cell cycle and oxidative damage. Due to the fragility of animal cells, it is unable to obtain further progress about the toxic effects of nanomaterials on cells so far. Physarum Polycephalum is an environmentally sensitive archaea, having better stress tolerance. In P.Physarum materials research for testing nano materials stress, we may get more details of the biological effects of nanomaterials and cellular stress response process.To determine the "turning point" dose to P.Physarum growth of different nanomaterials from stimulus to suppression, the paper observed the growth of Physarum plasmodium in the plate containing different concentrations of nm- TiO₂?nanometer- TiO₂?, nm-CuTiO₂?nanometer CuTiO₂, nano-CuTiO₂ 1031 E, nano-CuTiO₂ 1229 G, nano-CuTiO₂ 1225 G, three types of nano materials which copper content were 40%, 45%, 55%?, nm- Betaine?nanometer Betaine?. The growth of plasmodium would rise enhanced when the nm- TiO₂ concentration below 9 mg / m L, the growth of plasmodium had be suppressed when nm- TiO₂ concentration exceeding 15 mg / m L. The growth of plasmodium improved and enhanced with nm-CuTiO₂ concentration less than 0.3mg / m L. When the nm-CuTiO₂ concentration exceeding 0.5 mg / m L, the growth was suppressed as concentration of nm-CuTiO₂ increasing. nm-CuTiO₂ induced a dose-dependent suppressing growth of plasmodium by C u content. In culture plates containing 0.2mg / ml nm-betaine, the P.Physarum significant role in promoting growth, while significantly inhibited when concentrations exceeding 0.5 mg / m L. The results indicate that regardless of stress from inorganic or organic nano- materials, different concentration and types of nanomaterials may result in different effect.To explore the growth mechanisme of nanomaterials in P.Physarum, we used DCFH-DA to react with ROS?reactive oxygen species? in P.Physarum before they were exposed to nanomaterials, resulting in increased production of ROS with concentration rising.To understand the oxidative damage of nanomaterials stress on cell membra ne, we tested MDA content in P.Physarum cultivated on cultures composing of different concentrations and different types of nanomaterials. The results showed that below 0.2mg / ml nm-CuTiO₂ 1225 G, nm-CuTiO₂ 1229 G, nm-CuTiO₂ 1031 E and nm- Betaine, MDA level did not change significantly, indicating that low dose of nano materials stress couldn't cause cell membrane oxidative damage. With concentration of nanomaterials rising, nm-CuTiO₂ 1225 G, first reached the apex level of MDA and had been higher than other nanomaterials stress situation. The level of MDA in nm-CuTiO₂ 1229 G stress was inferior nm-CuTiO₂ 1225 G stress cases, but nm-CuTiO₂ 1031 E and organic nm- Betaine on MDA impact is not obvious, indicating that higher copper content caused stronger oxidative damage.To understand the stimulatory and inhibitory effects on gene expression of nm- TiO₂, nm-CuTiO₂ and nm-Betaine in P.Physarum growth, we started a high-throughput sequencing techniques to detect the transcriptome of P.Physarum in nm- TiO₂, nm-CuTiO₂ and nm-Betaine significantly stimulate and inhibit the growth, without any stress of the P.Physarum as a control. By comparing the transcriptome data analysis to find out the gene expression and the signal path affects of above- mentioned nanomaterials in cell growth stimulatory and inhibitory effects.The results showed that in 9 mg / m L nm- TiO₂, 193 significant differentially expressed genes were acquired?qvalue <0.005 and | log2 Fold C hange |> 1?, in which 127 up-regulated genes and 66 down-regulated genes. The mainly rich set in pyrimidine metabolism pathway?P-value = 0.045?, illustrating nm- TiO₂ stress may affect the synthesis of DNA. Under 15 mg / m L nm- TiO₂ stress, 392 significant differentially expressed genes were found, of which 312 up-regulated genes and 80 down-regulated genes, enriched in DNA replication?P-value = 4.82E-09?, the cell cycle?P-value = 1.07E-05? and DNA repair pathways.The result suggests the impact of cell replication by nm- TiO₂.The results showed that in 0.3mg / ml nm-CuTiO₂, 280 significantly differentially expressed genes were acquired, 179 up-regulated genes and 101 down-regulated genes. In 0.5mg / ml nm-CuTiO₂, a total of 1643 differentially expressed genes were found, of which 968 up-regulated genes and 675 down-regulated genes,these genes are involved in ribosome biogenesis?P-value = 1.53E-19?, DN A replication?P-value = 0.0143? and homologous recombination?P-value = 0.037200594?. The result indicates that nm-CuTiO₂ may have a certain influence on DNA replicated,protein synthesis in P.Physarum cells.The result showed that in 0.2mg / ml nm-betaine, 387 significantly differentially expressed genes were found, of which 309 up-regulated genes and 78 down-regulated genes. There were 374 differentially expressed genes in 0.5mg / ml nm-betaine stress, 286 up-regulated genes and 88 down-regulated genes.The differentially expressed genes mainly involved in DNA replication?P-value = 1.10E-12?, the cell cycle?P-value = 7.41E-08?,and base excision repair?P-value = 2.58E-06?etc. The results demonstrate that the nm-betaine stress can cause DNA damage.To understand the co-stimulatory and co- inhibitory effects on gene expression of nm- TiO₂, nm-CuTiO₂ and nm- Betaine in Physarum growth,we found 36 differentially co-expressed genes at high concentrations of nanomaterials stress and of 39 differentially co-expressed genes at low concentrations of nanomaterials stress by analysising the transcriptome results of P. Physarum. Verification by Real- Time PCR is consistent with the results obtained by transcriptome sequencing, indicating that transcriptome sequencing results are credible. These provide the foundation for the standards establishment of nanomaterials cytotoxicity and biological safety evaluation.