| Due to their excellent antibacterical activity,silver nanoparticles?Ag NPs?and its products have been extensively produced and widely used in the field of biological medicine,industrial production and daily life,and so on.The extensive applications of Ag NPs will inevitably cause Ag NPs release into the environment,especially for the aquatic environment.More and more researches have demonstrated that the Ag NPs in water environment system will enter the living organisms through a variety of channels?e.g.breath,ingestion and skin?and then transmit along the food chain,which has raised thepublic concern of its potential risks to environment and human health.Previous studies on the toxicity of Ag NPs features the long period and enormous amounts of experimental expense,and focus on the single model organism and the route of exposure.Once the organism has been died or the growth and reproduction are blocked,indicated that the ecological effects of Ag NPs have reached an irreversible stage.Generally,the effects of Ag NPs on ecosystem origined from the biomacromolecule in the organism?e.g.nucleotide and protein?,and then appeared gradually in the cell,tissue,individual,population,community and ecosystem,which have the greatest predictive value about the assessment of ecosystem.Therefore,the objective of the present study is to investigate the molecular toxicity of Ag NPs on a typical aquatic orgaisms namely,Scenedesmus obliquus and Daphnia magna,using advanced transcriptome technology.The main contents and conclusions can be listed as follows:?1?Synthesis and characterization of Ag NPs.In a typical top-down process,Ag NPs was freshly prepared by mixed aqueous solutions of L-glutathione reduced?GSH?and Ag NO3,followed by the addition of NaBH4.TEM images demonstrated that the Ag NPs were dispersive and had the size below 1.5nm.The solution of Ag NPs is light brown and show a strong UV-vis absorption of peak at 489nm.An intensive red fluorescence was emitted by Ag NPs with an excitation of 430 nm.The molecular structure of Ag NPs was assigned to Ag16?SG?9 by way of ESI mass spectra and MALDI-TOF mass spectra.?2?The acute toxicity of Ag NPs to Scenedesmus obliquus.After the algae cells were exposed to the increasing concentration of Ag NPs,and the results demonstrated that Ag NPs had obvious inhibitory effects on the growth of algae cells and also shown dose-effect relationships in the field of traditional toxicology.However,the contents of chlorophyl a of algae cells displayed the Low dose excitatory effect of hormesis rather than the dose-effect relationships.The concentration of reactive oxygen species?ROS?in algae cells was directly determined by electron paramagnetic resonance,indicating that the Ag NPs-mediated toxicity to algae cells was probably due to the joint-toxicity between Ag NPs and its dissolved Ag ions and was independent of ROS.Moreover,laser scanning confocal microscopy?LSCM?taking advantage of fluorescence characteristics of Ag NPs was employed to confirm the presence of Ag NPs in the algae cells.?3?Effect of Ag NPs and its dissolved Ag ions on Scenedesmus obliquus.The concentration of Ag ions dissolved from Ag NPs in pure water and SE medium were determined by ICP-Ms at 2,6,12,24,48,72 and 96 h,respectively.The dissolution experiments of Ag NPs indicated that the concentration range of Ag ions dissolved from Ag NPs in SE medium??g/L?was approximately equivalent to that in pure water.After that,algae cells?5.010.0×105 cells/mL?were exposed for 96h to the control,Ag NPs?135?g/L?,Ag NPs?135?g/L,with L-cysteine?,and Ag ions?AgNO3,5.0,10.0 and 20.0?g/L?,and the results demonstrated that the Ag NPs-mediated toxicity to algae cells was attributed to the joint-toxicity between Ag NPs and its released Ag ions and was independent of ROS.Moreover,Ag NPs and its dissolved Ag ions have apparent inhibition effects on the contents of chlorophyl a of algae cells in the early stages of exposure experiment,indicating that photosynthesis parameters,such as chlorophyl a,can be used as biomarker to the early diagnosis of the ecological risks of Ag NPs.However,this type of inhibition effects also shown the Low dose excitatory effect of hormesis rather than the dose-effect relationships.As a results,the phototoxicity of Ag NPs and its dissolved Ag ions on algae cells should be further investigated at the molecular level.?4?The phototoxicity of Ag NPs and its dissolved Ag ions on Scenedesmus obliquus.After algae cells?5.010.0×105 cells/mL?were exposed for 96h to the control,Ag NPs?135?g/L?,Ag NPs?135?g/L,with L-cysteine?,and Ag ions?AgNO3,5.0,10.0 and 20.0?g/L?,the photosynthesis parameters of algae cells,such as,??Utilization efficiency of light energy?,Fv/Fm?Photosynthetic Yield?PSII?=?Fm–F0?/Fm?,Ik?Saturated light intensity?and rETRMax?Maximum relative electron transport rate?,were determined by a PHYTO-PAM Phytoplankton Analyzer at 24,48,72 and 96 h,respectively.The fluorescence intensity of algae cells exposed to Ag NPs and its dissolved Ag ions was then determined by laser scanning confocal microscopy basing on the fluorescence characteristics of Ag NPs,which further conformed that Ag NPs will enter the algae cells.Finally,the photosynthesis toxicity of Ag NPs and its dissolved Ag ions on algae were characterized by transcriptome technique,and the results further conformed that the Ag NPs-mediated toxicity to algae cells was due to the joint-toxicity between Ag NPs and its dissolved Ag ions.In addition,several key genes,such as psbA,petF and ATPF0C encoded for the reaction center complexes proteins of the light reaction of photosynthesis were down regulated as compared to the control,which further disturebed normal electron transfer of photoreaction and photophosphorylation process of ATP production.Meanwhile,the expression of key gene?e.g.rbcs?,encoding for the key enzymes-RuBP carboxylase/oxygenase?Rubisco?,was also down regulated and lead to the inhibition to the Calvin cycle.What is more,relevant down-regulated genes of the photosynthesis of algae cells mentioned above were also demonstrated by qRT-PCR experiment.?5?Genotoxic responses of Daphnia magna fed with green algae exposed to silver nanoclusters.First of all,Scenedesmus obliquus?5.010.0×105 cells/mL?were exposed for 96h to the control,Ag NPs?135?g/L?,Ag NPs?135?g/L,with L-cysteine?,and Ag ions?AgNO3,5.0,10.0 and 20.0?g/L?.After exposure for 96h to Ag,algae cells were filterd with 0.45?m cellulose acetate membrane for feeding to Daphnia magna.Advanced transcriptome technique was then employed to investigate to genotoxic responses of Daphnia magna fed with green algae exposed to silver nanoclusters.The results demonstrated that Ag NPs and its dissolved Ag ions have played synergetic roles in genotoxic responses of Daphnia magna fed with green algae.Moreover,key gene,such as PLA2G?SPLA2?encoded for secretory phospholipase A2,was down-regulated as compared to the control,which further inhibited the digestive function and glycerophospholipid metabolism of Daphnia magna.Our study suggested that the toxicity of Ag NPs and its dissolved Ag ions to living organisms origined from the macromolecule or cells,and then gradually emerged in tissues,organs andorganisms.What is more,advanced transcriptome technique exhibited unique properties,such as large scale and high throughput,and also made contribution to investigating the toxicity mechanisms at the molecular level and early-diagosis of ecological risks of Ag NPs. |
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