Nt cells. This sets a limit for the concentrations to be used when carrying out experiments on plants using nanoparticles of this type. As previously reported [8], when in contact with the plant cell suspensions, some nanoparticle aggregation was observed. At 10 nM this occurrence is small, but is amplified at higher concentrations. Aggregation may mask an even higher level of stress caused by these nanoparticles at higher concentrations than 10 nM, preventing their absorption into cells. M. sativa cells responded to the oxidative stress caused by the addition of MPA-CdSe/ZnS QD by activating their antioxidant enzyme systems. In this study, three antioxidant enzymes: SOD, CAT and GR were activated within 48 hours of MPA-CdSe/ZnS QD exposure, preventing over-accumulation of H2O2 and O2?? as shown previously [8]. Higher concentrations of MPACdSe/ZnS QD may induce the accumulation of ROS that are able to damage the plasma membrane, mitochondria and nucleus. Cells adapt to the imposed stress by up-regulating antioxidant and/or repair systems. This may protect them against damage to some extent, or sometimes even overprotect them; the cells are then resistant to higher levels of oxidative stress imposed subsequently [36]. This is the first report on the genotoxic effects of MPA-CdSe/ZnS QD in plant cells and demonstrates that both the DNA repair genes (Tdp1, Top1 and FPG) and the ROS scavenging mechanisms are activated when these QD interacts with M. sativa cells. MethodsSynthesis and characterization of QDnanoparticles are exerting a genotoxic effect that the cells try to counteract by increasing the expression of these genes. This is corroborated by the data obtained from the Comet assays, that show that even 10 nM of MPA-CdSe/ZnS QD may induce a genotoxic response by plant cells. The fact that the expression of APX and SOD genes is also up-regulated by the nanoparticles (Figure 4), mostly at the highest concentrations, is in3-Mercaptopropanoic acid coated CdSe/ZnS QD were synthesized, solubilised and characterised according to Miguel et al. [5]. In brief, MPA-CdSe/ZnS QD were obtained by the phase transfer method and the resultant water-soluble QD were purified and concentrated using a Sartorius Vivaspin 6 tube (cut-off 10KDa) at 7500 g. For the characterisation of the synthesized CdSe/ZnS core-shell QD, Transmission Electron Microscopy (TEM)Santos et al. BMC Biotechnology 2013, 13:111 http://www.biomedcentral.com/1472-6750/13/Page 7 ofwas used. Low-resolution Stattic supplement images were obtained using a JEOL 200CX traditional TEM operating at an acceleration voltage of 200 kV. Dynamic Light Scattering (DLS) analysis was LDN193189 dose performed using a Zetasizer Nano ZS dynamic light scatterer from Malvern Instruments. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27532042 The watersoluble QD had a hydrodynamic diameter of 13.5 nm and zeta potential of -46.5 mV. The concentration of the stock solution was determined as in [5] using the spectrophotometric method of Yu et al. [37,38]. Appropriate dilution of this stock solution afforded the solutions used in this study.Cell suspension culture treatments(10 seconds interval) in a 1 mL solution containing 0.5 mM xanthine, 0.05 mM ferricytochrome-C, 0.1 mM EDTA, 0.01U of xanthine-oxidase and 0.05 mL of enzyme extract in 100 mM potassium phosphate buffer (pH 7.5). The enzymatic activity was estimated as the quantity of enzyme necessary for the inhibition of 50 of ferricytochrome-C reduction per minute under the assay conditions [41]: Units=mg protein ?? inhibition=50 ? 1=v g.Nt cells. This sets a limit for the concentrations to be used when carrying out experiments on plants using nanoparticles of this type. As previously reported [8], when in contact with the plant cell suspensions, some nanoparticle aggregation was observed. At 10 nM this occurrence is small, but is amplified at higher concentrations. Aggregation may mask an even higher level of stress caused by these nanoparticles at higher concentrations than 10 nM, preventing their absorption into cells. M. sativa cells responded to the oxidative stress caused by the addition of MPA-CdSe/ZnS QD by activating their antioxidant enzyme systems. In this study, three antioxidant enzymes: SOD, CAT and GR were activated within 48 hours of MPA-CdSe/ZnS QD exposure, preventing over-accumulation of H2O2 and O2?? as shown previously [8]. Higher concentrations of MPACdSe/ZnS QD may induce the accumulation of ROS that are able to damage the plasma membrane, mitochondria and nucleus. Cells adapt to the imposed stress by up-regulating antioxidant and/or repair systems. This may protect them against damage to some extent, or sometimes even overprotect them; the cells are then resistant to higher levels of oxidative stress imposed subsequently [36]. This is the first report on the genotoxic effects of MPA-CdSe/ZnS QD in plant cells and demonstrates that both the DNA repair genes (Tdp1, Top1 and FPG) and the ROS scavenging mechanisms are activated when these QD interacts with M. sativa cells. MethodsSynthesis and characterization of QDnanoparticles are exerting a genotoxic effect that the cells try to counteract by increasing the expression of these genes. This is corroborated by the data obtained from the Comet assays, that show that even 10 nM of MPA-CdSe/ZnS QD may induce a genotoxic response by plant cells. The fact that the expression of APX and SOD genes is also up-regulated by the nanoparticles (Figure 4), mostly at the highest concentrations, is in3-Mercaptopropanoic acid coated CdSe/ZnS QD were synthesized, solubilised and characterised according to Miguel et al. [5]. In brief, MPA-CdSe/ZnS QD were obtained by the phase transfer method and the resultant water-soluble QD were purified and concentrated using a Sartorius Vivaspin 6 tube (cut-off 10KDa) at 7500 g. For the characterisation of the synthesized CdSe/ZnS core-shell QD, Transmission Electron Microscopy (TEM)Santos et al. BMC Biotechnology 2013, 13:111 http://www.biomedcentral.com/1472-6750/13/Page 7 ofwas used. Low-resolution images were obtained using a JEOL 200CX traditional TEM operating at an acceleration voltage of 200 kV. Dynamic Light Scattering (DLS) analysis was performed using a Zetasizer Nano ZS dynamic light scatterer from Malvern Instruments. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27532042 The watersoluble QD had a hydrodynamic diameter of 13.5 nm and zeta potential of -46.5 mV. The concentration of the stock solution was determined as in [5] using the spectrophotometric method of Yu et al. [37,38]. Appropriate dilution of this stock solution afforded the solutions used in this study.Cell suspension culture treatments(10 seconds interval) in a 1 mL solution containing 0.5 mM xanthine, 0.05 mM ferricytochrome-C, 0.1 mM EDTA, 0.01U of xanthine-oxidase and 0.05 mL of enzyme extract in 100 mM potassium phosphate buffer (pH 7.5). The enzymatic activity was estimated as the quantity of enzyme necessary for the inhibition of 50 of ferricytochrome-C reduction per minute under the assay conditions [41]: Units=mg protein ?? inhibition=50 ? 1=v g.