nanoparticles are being 
 introduced , in some cases rapidly , into 
 the consumer market but there is a fundamental lack of knowledge on 
 their potential consequences for human and environmental health ( reviewed 
 in refs ( 1 and 2 ) ) .
the use of silver 
 nanoparticles ( agnp ) has increased very significantly in recent years , 
 with a current total global production estimated at 500 tonnes per 
 year ( reviewed in ref ( 1 ) ) .
very little empirical data are available on environmental levels 
 of agnp but given their very significant production , it is likely 
 that agnp are reaching the aquatic environment where they may impact 
 on living organisms , as either particulates and/or as dissolved silver 
 ions .
the susceptibility of freshwater fish to the toxic effects 
 of exposure 
 to silver ions ( ag ) is well documented and acute toxicity 
 is associated predominantly with ionic ag interactions 
 at the gills , where they inhibit basolateral na / k - atpase activity .
the toxicity of this metal contaminant is affected by ag speciation , 
 which in turn is affected by a number of parameters including ionic 
 composition and organic matter dissolved in the water . as a consequence 
 of the decreased bioavailability of ag with increased ionic strength 
 of the water , fish inhabiting freshwater environments are more susceptible 
 to ag toxicity compared with marine fish species ( reviewed in ref ( 6 ) ) . a number of studies 
 investigating the toxicity of agnp , in a range 
 of fish species ,
have provided evidence for concentration - dependent 
 toxicity ( reviewed in ref ( 1 ) ) .
agnp have been shown to enter zebrafish embryos via the 
 chorion pore canals and were reported to be present in the brain , 
 heart , yolk and blood of developing zebrafish . in 
 the study by asharani and colleagues ,
however , 
 exposures to agnp were conducted at very high concentrations and the 
 particles had been stabilized with starch or bsa to prevent agglomeration . 

the conditions adopted were likely to enhance uptake in those laboratory 
 exposures , but they may not reflect environmental reality . in juvenile 
 or adult fish ,
one of the main routes of uptake and toxicity for agnp 
 appears to be via the gills , and adverse effects include on osmoregulation , 
 similar to that reported for ionic silver . for 
 agnp ,
the effect on na / k - atpase activity in 
 gills has been attributed to the dissolution of silver ions from the 
 particles , and/or to the presence of silver ions at the surface of 
 the agnp .
agnp have also been reported 
 to reduce the ability of fish to extract oxygen from the water during 
 progressive oxygen depletion , without 
 causing morphological alterations on the gill .
the 
 mechanisms by which this specific effect of exposure to agnp occurs 
 are unclear and may be due to damage of the gill surface caused by 
 the presence of particulate matter and/or local dissociation of ag from the agnp trapped in the mucus surrounding the gill membrane . 

the biological processes affected by exposure to agnp , reported across 
 a variety of organs , include induction of oxidative damage , alterations 
 to the regulation of enzymes responsible for free radical scavenging , 
 altered regulation of gene expression pathways involved in apoptosis , 
 and disrupted regulation of the cellular machinery involved in storing , 
 detoxification and metabolism of metals ( such as metallothionein 2 ) .
the objective of this study was to investigate the molecular 
 mechanisms 
 of toxicity of agnp ( 10 nm ) in comparison with a micrometer sized 
 counterpart ( ag bulk , 0.6 - 1.6 m ) and with ag , using 
 a recently developed high - throughput ( ht-)supersage approach on an 
 illumina ga ii platform and zebrafish 
 ( embryos ) as a model organism .
our data demonstrated that most transcriptomic 
 alterations caused by exposure to agnp and its ionic and bulk counterparts 
 were common across all treatments , supporting the hypothesis that 
 the toxicity of agnp is principally associated with the toxicity of 
 ag at the surface of the particles or dissolved in the 
 water .
we also identified some gene changes unique to each treatment , 
 suggesting that there may also be some particle - specific effects .
silver particles ( designated as agnp [ 10 nm ] , and ag 
 bulk [ 0.61.6 m ] ) were purchased from nanostructured 
 and amorphous materials inc . ,
houston , tx and silver with 2% hno3 was obtained from perkinelmer life and analytical sciences , 
 shelton , ct .
further details on the characterization of these particles 
 and their dissolution in exposure water can be found in ref ( 13 ) and in the supporting information ( si ) .
adult zebrafish 
 ( wild - type wik strain , originally from the max planck institute , tubingen , 
 germany ) for the provision of embryos were kept in aquaria at the 
 university of exeter in 140 l mixed - sex stock tanks , as described 
 in ref ( 18 ) .
fish were 
 allowed to breed naturally and eggs were collected in glass egg chambers , 
 approximately 1 h postfertilization ( hpf ) .
stock solutions for agnp , ag bulk 
 and silver nitrate were made up in ultrapure water , and sonicated 
 for 1 h to ensure dispersal of the particles .
exposures were conducted 
 in glass chambers , at 28  1 c with a 12 h light : dark photoperiod . 
 immediately prior to the start of the exposures , glass chambers received 
 400 ml artificial water , prepared according to the iso
73463:1996 
 guideline , containing 5 g / l of 
 10 nm agnp , 5 g / l of ag bulk or 0.25 g / l of silver nitrate . 

these exposure 
 concentrations were well below those causing overt toxicity , based 
 on the mortality curves for the various forms of silver obtained under 
 the same experimental conditions in our laboratory ( see si figure s1 ) .
the dosing of ag bulk was chosen 
 to be the same as that for the agnp ( in terms of mass ) , to allow us 
 to test whether the size of the particles was a determinant of the 
 effects seen .
g / l was 
 included to provide a likely worst case scenario for the highest dissolution 
 of ag for the agnp and ag bulk exposures in the adopted 
 exposure conditions , based on data from the literature suggesting 
 that dissolution of agnp is variable but below 5% ( osborne et al 2012 
 reported this to vary between 0.1 and 2% in zebrafish embryo culture 
 water ( 20 ) ) .
our own experiments ( see si ) suggest that dissolution rates for agnp and 
 ag bulk under our experimental conditions were 0.27%  0.15% 
 and 0.27%  0.07% , respectively .
exposures were initiated at 
 4 hpf and 585  99 eggs were deployed into each treatment chamber . 

unfertilized eggs were identified by visual inspection using light 
 microscopy ( kyowa optical sdz pl , kyowa optical , japan ) and removed 
 from the exposure chamber ( 22.8% of the total number of eggs were 
 unfertilized ) .
solutions were replaced every 12 h during the exposure 
 period , and dead embryos were removed at the times of replacement 
 of the exposure water . at 24 h and 48 h , three pools of 50 embryos 

were removed from each of the exposure tanks , immediately frozen in 
 liquid nitrogen and stored at 80 c for analysis of gene 
 expression .
the experiment was terminated at 48 hpf . in order 
 to verify the biological significance of the effect of all treatments 
 on the oxidative phosphorylation pathway , we designed a study to evaluate 
 the effects of exposure to a series of doses of ag on 
 embryo oxygen consumption .
zebrafish embryos were exposed to a range 
 of concentrations of ag to include those expected to be 
 present in the water following exposure to agnp , ag bulk and ag ( 0 , 0.031 , 0.062 , 0.125 , 0.250 , 0.500 , 1 , and 5 g 

exposures were initiated at 4 hpf and maintained 
 for 48 h , and the average oxygen consumption per embryo was determined 
 for two 24 h periods ( 024 h and 2448 h from the onset 
 of the exposure ) .
the full description of the methodology adopted 
 for these procedures is given in the si .
rna 
 extraction was conducted on each pool of 50 embryos using the 
 rnaeasy kit , according to the manufacturer s instructions ( qiagen , 
 uk ) .
the quality and concentration of the resulting rna were determined 
 using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , 
 wilmington , de ) , and an agilent bioanalyser 2100 ( agilent technologies , 
 inc . ,
ht - supersage was conducted according to previously 
 described methods and libraries were 
 sequenced using an illumina genome analyzer ( ga ) ii platform .
further 
 details are given in the si . in total , 
 over 16.9 million 
 36-bp sequences were obtained and processed , using the supersage data 
 analysis procedure presented in figure 1 .
a 
 selection of tools from the fast - toolkit ( http://hannonlab.cshl.edu/fastx_toolkit/ ) and our own custom perl scripts were used , running under the nebc 
 bio - linux environment .
the fastq / a barcode 
 splitter ( fastx - toolkit ) was used to separate the samples from each 
 lane based on the 4 base barcodes .
the barcodes were removed from 
 the sequences using the fastq / a trimmer ( fastx - toolkit ) , before converting 
 the sequences to fasta format using fastq - to - fasta ( fastx - toolkit ) . 

a perl script ( si , file s1 ) was used to 
 remove all remaining adapter sequences after the last occurrence of 
 catg ( nlaiii restriction site , used for sequence 
 tag preparation ) in each of the sequences .
fastx_collapser ( fastx - toolkit ) 
 was used to collapse the sequences and calculate the frequency of 
 unique sequence tags ( unitags ) in each library .
unitags 
 were mapped to all available zebrafish genes ( 28  491 genes ) 
 in ensembl zv9 ( release 60 ; ( 22 ) ) using bowtie , with no mismatches allowed .
counts of unitags 
 that mapped to the same gene were summed and unitags that aligned 
 with more than one gene were removed from the data set using the perl 
 scripts provided in si file s2 . for each 
 treatment , the counts of unitags representing each ( unique ) gene were 
 normalized to 1 million tags .
fold changes ( fc ) were determined for 
 each gene by dividing the number of tags in the normalized silver 
 treatment libraries by the number of tags in the normalized control 
 libraries .
fc values less than 1 ( down - regulated genes ) were transformed 
 using the following formula : 1/fc , in order to center the 
 fold change values around 0 .
differences in gene expression between 
 the silver treatments and the controls were determined using discoveryspace 
 4.0 , which implements the audic - claverie 
 significance test to account for differences in sample size and p values were adjusted using a false discovery 
 rate correction .
genes that were found 
 to be significantly altered as a result of the silver treatments ( adjusted p < 0.05 ) were further investigated . in order to visualize 
 differences and similarities in gene expression between each of the 
 silver treatments and the controls ,
scatter plots were produced using 
 discoveryspace and correlation analysis ( pearson s ) was performed 
 using sigmaplot 11 ( systat software uk ltd . ) .
functional annotation analysis 
 was performed on the lists of differentially 
 expressed genes ( adjusted p < 0.05 ) for each treatment 
 using the database for annotation , visualization and integrated discovery 
 ( david v6.7 ; ) and using all genes that were 
 found to be expressed in zebrafish embryos at 24 hpf ( 12  939 
 genes ) or 48 hpf ( 13  584 genes ) , respectively , as background . 

gene ontologies ( go ) for biological processes , cellular components 
 and molecular function were considered significantly enriched when p < 0.05 .
pathway analysis 
 was conducted through 
 the use of ingenuity pathways analysis ( ipa ; ingenuity systems , http://www.ingenuity.com ) , based on the lists of differentially 
 expressed genes ( adjusted p < 0.05 ) for each of 
 the treatments .
enrichment of canonical pathways was determined using 
 the ipa library of canonical pathways , with a p value cutoff of 0.05 
 ( adjusted for multiple testing using the fisher s exact test ) . 

similarly , enriched kegg pathways were determined using the lists 
 of differentially expressed genes ( adjusted p < 
 0.05 ) and all expressed genes at 24 hpf and 48 hpf , respectively , 
 as background using david .
statistical 
 analysis of 
 the data for embryo survival and oxygen consumption were conducted 
 within sigmaplot 11 ( systat software uk ltd . ) .
changes in embryo survival 
 caused by exposure to agnp , ag bulk or ag during the exposure 
 experiments for molecular analysis were analyzed using chi - square 
 tests .
relationships between the concentration of ag and 
 oxygen consumption were investigated using regression analysis .
differences 
 between treatment groups were determined using one way analysis of 
 variance ( anova ) , followed by an all pair - wise multiple comparison 
 procedure ( student  newman  keuls method ) . for these tests , 

data were considered to be significant when p < 
 0.05 . the raw reads and processed unitag 
 frequency tables for each of the ht - supersage libraries have been 
 deposited in ncbi s gene expression omnibus and are accessible through geo series accession number 
 gse38125 .
embryo mortality 
 during the course 
 of the exposures for generation of embryo samples for molecular analysis 
 was 4.6  2.5% , which falls within that expected for zebrafish 
 and within the guidelines for oecd standardized testing guidelines .
there were no differences in embryo mortality 
 rates between treatment groups ( p = 0.213 ) , confirming 
 that all exposures were conducted at concentrations below those causing 
 any overt toxicity .
the number 
 of sequences 
 obtained for each library ranged from 1.2 million ( 48 hpf agnp ) to 
 2.2 million ( 24 hpf agnp ) and the abundance distributions of these 
 sequence tags were generally consistent between all libraries ( si table s1 ) . in total , 13  680  994 
 sequences were taken further for analysis and these were represented 
 by 606  259 unique sequence tags ( unitags ) . in total 99  683 
 unitags mapped to zebrafish genes , with 77  821 of these mapping 
 to unique genes only ( 7  690  269 counts in total ) and 
 representing 14  793 genes ( figure 1 ) . 

this is consistent with previous studies that reported a similar number 
 of expressed genes in embryos at similar stages of development using 
 zebrafish affymetrix microarrays ( arrayexpress database [ http://www.ebi.ac.uk/arrayexpress/ ] ; experiment i d : e - tabm-33 ) or rna - seq .
approximately 80% of the genes were represented by more than one 
 unitag ( see si figure s2 ) , consistent with 
 other studies that are based on sage methods , which is likely the 
 result of incomplete nlaiii digestion during the 
 library preparations , alternative polyadenylation and/or alternative 
 splicing sites .
statistical analysis of the gene expression 
 data identified genes differentially expressed between the different 
 treatments and the control , for both 24 hpf and 48 hpf ( summarized 
 in si file s3 ) .
a visual representation 
 of the expression profiles of each treatment group compared to the 
 respective control is presented in si figure 
 s3 .
there was a strong correlation between the levels of expression 
 of genes in the controls compared with those in each treatment group 
 ( r > 0.937 ) .
the number of differentially expressed 
 genes per treatment group , and the overlap between the differentially 
 expressed genes for each treatment were consistent across all treatments , 
 at both 24 h and 48 h of exposure ( with the exception of the treatment 
 24 h ag ; which showed a greater proportion of differentially 
 expressed genes ; figures 2a , b , and c ) .
a heatmap 
 of the differentially expressed genes across the various silver treatment 
 libraries is shown in si figure s4 , and 
 demonstrates the consistency in gene expression across all treatments . 

the condition tree highlights the differences in gene expression profiles 
 for the differentially expressed genes between the 24 h ag compared to all other treatments and between embryos collected at 
 24 hpf and 48 hpf .
( a ) . the number of differentially expressed genes ( adjusted p values < 0.05 ) as a result of the various silver treatments 
 ( agnp , ag bulk and ag ) compared to the controls . percentages 
 of up- and down - regulated genes for each of the silver treatments 
 are indicated on the bars .
venn - diagram showing the overlap 
 between sets of differentially expressed genes obtained for each of 
 the silver treatment libraries after 24 ( b ) and 48 ( c ) hours of exposure .
some of the differentially expressed 
 genes were found to be specific 
 to each of the different treatments ( agnp  13 genes ; ag bulk 
 1 gene ; ag - 32 genes ) , reflecting gene responses 
 distinct to the different treatment ( si table s2 ) .
distinct genes were found to be differentially expressed 
 at both time points for any one treatment and not present in the list 
 of differentially expressed genes for the other treatments at any 
 time point .
we explored 
 the biological significance of the changes in gene expression caused 
 by exposure to agnp , ag bulk and ag in 24 hpf and 48 hpf 
 embryos by determining the enrichment in go terms and canonical pathways 
 using david and ipa .
analyses were conducted based on differentially 
 expressed genes , including all up- and down - regulated genes , to obtain 
 a global overview of the biological effects of the three distinct 
 treatments ( figure 3 , si figure s5 and file s5 ) .
the most significantly over - represented 
 go terms and kegg pathways ( ribosome and oxidative phosphorylation ) 
 were common to all three treatments within each time of sampling , 
 demonstrating a very significant overlap in the mechanisms of toxicity 
 of the three forms or silver considered in this study . based on this 
 overlap
, it seems likely that the toxicity of agnp and ag bulk to 
 zebrafish embryos is predominantly associated with the toxicity of 
 free ag .
kegg pathways over - represented in differentially expressed 
 gene 
 lists following exposure of zebrafish embryos to silver . over - representation 

analysis was conducted for gene lists differentially expressed between 
 each treatment and its control ( adjusted p values 
 < 0.05 ) , after 24 and 48 h of exposure , using the list of all expressed 
 genes as a background , within david .
the full data set for kegg pathway 
 over - representation is presented in si , 
 file s5 .
the dissolution rates of agnp 
 and ag bulk in our experiments were 
 0.27%  0.15% and 0.27%  0.07% , respectively .
parallel 
 studies in our laboratory , investigating the dissolution rates for 
 agnp and bulk silver in embryo culture water , have found them to vary 
 between 0.1 and 2% over a 48 h period .
furthermore , very recently published data suggest that some silver 
 particles ( 41.6  9.1 nm agnps at a concentration of 0.2 nm ) 
 can penetrate the chorion through diffusion via the chorion canals and that once inside the chorion , the dissolution 
 rate can be much higher compared to that observed in the exposure 
 water .
consequently , the amount of bioavailable 
 ag in the agnp and ag bulk treatments would likely be 
 sufficient to cause most of the changes in gene expression observed . 
 for many of the genes significantly affected in all the exposures , 

the changes were more pronounced ( in terms of fold - change ) for the 
 exposure to ag compared with those observed for agnp and 
 ag bulk , and this is likely to be associated , at least in part , with 
 the bioavailability of silver ions in each treatment .
however , the 
 pathways affected ( as assessed by the analysis of go and kegg pathway 
 enrichment ( figure 3 , si figure s5 and file s5 ) ) remained common across all treatments .
these 
 changes are consistent with previous literature reporting effects 
 of exposure to ag ( ions or nanoparticles ) on several biological processes 
 across a series of model organisms .
the 
 most significantly enriched go terms for all treatments , both 
 at 24 and 48 hpf were ribosome , translation and structural constituents 
 of ribosome , for cellular components , molecular functions and biological 
 processes , respectively .
concurrently , the most enriched kegg pathway 
 was ribosome ( figure 3 and si file s5 ) .
these results align well with previous literature 
 that report disruption to protein biosynthesis pathways in a range 
 of organisms following exposure to agnp and ag .
for example , rpl10a was down - regulated 
 by 5.6 , 3.4 and 5.6-fold ( 24 h ) , and rpl18a down - regulated 
 by 12.2 , 1.2 and 9.7-fold ( 48 h ) , following exposure to agnp , ag bulk , 
 and ag , respectively .
expression of several genes encoding 
 components of the ubiquitin proteasome system were also altered across 
 all treatments , ( at 24 and 48 h ) , indicating that protein degradation 
 pathways were also altered by the treatments ( si file s3 ) .
oxidative phosphorylation was affected 
 consistently by the exposure 
 to agnp , ag bulk , and ag at both time points analyzed 
 ( figure 3 ) .
this is a complex pathway that 
 plays a key role in atp generation in aerobic organisms , and is comprised 
 of a series of five complexes and other associated molecules that 
 act in concert within the mitochondrial inner membrane .
the gene expression 
 profiles following 24 h of exposure to all forms of ag showed a down - regulation 
 of many of the genes encoding for complex i , ii , iii and iv ( figure 4 and si file s6 ) .
within 
 the complexes some genes were up - regulated whereas others were down - regulated , 
 in particular for complex i ( for ag and ag bulk ) .
this 
 complex is comprised of a large number of genes that form 14 central 
 and 32 accessory subunits , encoded by both the mitochondrial and the 
 nuclear genome , and that associate into three functional modules , 
 that oxidize nadh , reduce quinone and pump protons across the membrane 
 ( reviewed in ref ( 39 ) ) .
our data may reflect a differential regulation of the various 
 subunits and/or a differential regulation of the various functions 
 of complex i. overall , following 24 h of exposure to all ag treatments , 
 the gene expression data indicated a down - regulation of all complexes 
 involved in oxidative phosphorylation , possibly with the exception 
 of complex i for ag bulk ( figure 4 and si file s6 ) .
this effect is consistent with previously 
 reported observations reporting a down - regulation of this pathway 
 following ag exposure in a human cell line , using biochemical methods .
the degree to which these genes were regulated 
 between the various treatments was consistent with predicted differences 
 in the concentrations of bioavailable ag , which were expected 
 to be highest for ag , followed by agnp and lowest for 
 ag bulk .
effects of the silver treatments on transcription of genes belonging 
 to the mitochondrial dysfunction pathway .
shades of green and red 
 represent down - regulation and up - regulation of target genes , respectively . 

( a ) effects of exposure to agnp for 24 h on the mitochondrial dysfunction 
 pathway .
( b ) summary of the effects of exposure to all treatments 
 on the mitochondrial dysfunction pathway for 24 h and 48 h. numbers 
 in the table represent fold - change compared to the time - matched control . 

illustrations of the mitochondrial dysfunction and oxidative phosphorylation 
 pathways for all six treatments are presented in si , file s6 .
pathways were generated through the use of ingenuity 
 pathways analysis ( ingenuity systems , www.ingenuity.com ) .
data for embryos analyzed following 
 48 h of exposure to all ag 
 treatments similarly showed significant alterations in the expression 
 of genes encoding for proteins belonging to all complexes of the oxidative 
 phosphorylation pathway . when taken together ,
the changes in gene 
 expression suggest an up - regulation of these complexes , consistent 
 with a recovery of the activity in this pathway , during the 2448 
 h period , in exposed embryos ( figure 4 and si file s5 ) .
the changes in gene expression 
 for constituents of the oxidative 
 phosphorylation pathway suggested that ag may cause a down - regulation 
 in metabolic rates and atp synthesis during the first 24 h of exposure , 
 with a subsequent recovery from this effect at 48 h for all treatments . 

to further investigate the feasibility of this hypothesis , we conducted 
 an experiment to investigate the effects of ag on the 
 metabolic activity , measuring oxygen consumption as a proxy for aerobic 
 respiration of exposed embryos .
our data demonstrated that ag caused a decrease in oxygen consumption during the first 
 24 h of exposure ( r = 0.469 ; p < 0.001 ; figure 5 ) , with significant decreases 
 in oxygen consumption in embryos exposed to 0.125 , 0.5 , and 5 g 
 ag / l compared with those exposed to 0.031 g ag /
l , and 5 g ag / l compared to the controls 
 during the first 24 h of exposure ( one way anova followed by student  newman  keuls 
 method ; figure 5a ) .
this effect of ag on embryo oxygen consumption was no longer apparent for the second 
 24 h period ( between 24 and 48 h of exposure ( r = 
 0.114 ; p = 0.467 ; figure 5b ) , supporting the inhibitory effects of ag on 
 oxidative phosphorylation ( and associated decrease in oxygen consumption ) 
 during the first 24 h of exposure and subsequent recovery by 48 h. 
 together , these data support previous studies that have reported inhibition 
 of oxidative phosphorylation following exposure to silver nanoparticles 
 and silver ions in a range of mammalian model systems .
importantly , our data suggest that zebrafish embryos have the capacity 
 to deploy compensatory mechanisms at concentrations of ag below those 
 causing overt toxicity .
groups of 5 embryos were exposed to seven concentrations of 
 silver ions ( 05 g
/ l ) in isolated glass vials , and the 
 oxygen consumption was measured for 024h ( a ) and 2448 
 h ( b ) exposure periods ( n = six groups of five embryos 
 per treatment ) .
there was a significant effect of the exposure to silver 
 ions for the 024 h , but not for the 2448 h exposure 
 periods ( one way anova , p < 0.001 , followed by 
 an all pairwise comparison between the groups using the student  newman 
letters above the box plots 
 represent statistical significance , with treatments with different 
 letters being significantly different from each other .
the mechanisms by which exposure to ag caused the 
 observed changes 
 in the oxidative phosphorylation pathway are difficult to ascertain . 

recent evidence from mammalian studies has shown that pyruvate kinase , 
 m2 ( pkm2 ) that catalyzes the rate limiting atp producing step of glycolysis , 
 plays a key role in maintaining cellular redox homeostasis in cancer 
 cells .
this enzyme is specifically oxidized 
 by h2o2 , resulting in a decrease of its activity , 
 decreased pyruvate formation and increased flux of glycolytic metabolites 
 into the pentose phosphate pathway .
this pathway produces reduced 
 nadph , a crucial source of reducing equivalents for fatty acid synthesis 
 and for the glutathione , peroxiredoxin and thioredoxin systems , which 
 play a key role in the detoxification of reactive oxygen species ( ros ) , 
 providing cancer cells with a protective mechanism from intracellular 
 ros ( reviewed in ref ( 45 ) ) . in contrast , normal mammalian cells express another pyruvate kinase 
 isoform ( pkm1 ) , which is not susceptible to oxidation by hydrogen 
 peroxide .
interestingly , we did not see expression of this later isoform 
 in either 24 h or 48 h embryos . instead , zebrafish embryos expressed 
 two forms of the h2o2 sensitive isoform ( pkm2a and pkmb2 ) .
the principal form of 
 pkm measured was pkm2a ( the average counts per million 
 in our data set were 226.0  14.0 for embryos analyzed at 24 
 h and 565.5  25.4 for embryos analyzed at 48 h ) .
pkm2b was also expressed , but at much lower levels ( 0.9  0.6 for 
 24 h embryos and 40.4  8.4 for 48 h embryos ) .
the fact that 
 both the expressed forms of pkm in zebrafish are 
 homologues to the human h2o2 sensitive form 
 suggest that if the intracellular levels of h2o2 increase , zebrafish embryos may have the capacity to partially switch 
 to the pentose phosphate pathway , as a mechanism of response to ros . 

increase in ros is likely to occur during exposure to ag , as demonstrated 
 by the increase of gene markers for oxidative stress in our study , 
 and in a number of other studies .
one of the most significant changes in gene expression across the 
 treatments was for alcohol dehydrogenase class-3 ( adh5 ) , which was 70.5 , 7.1 , and 125.0 fold up - regulated following exposure 
 to agnp , ag bulk , and ag , respectively , at 24 h. similarly , 
 after 48 h of exposure , adh5 was up - regulated by 
 151.0 , 22.8 , and 50.5 , as a result of exposure to agnp , ag bulk , and 
 ag , respectively .
alcohol dehydrogenases ( adh ) metabolize 
 ethanol and other alcohols and aldehydes . in mammals , there are several 
 classes of adh , and class i adh are responsible for the metabolism 
 of ethanol and other small chain alcohols
. class ii adh enzymes preferentially 
 metabolize larger alcohols and aldehydes ( reviewed in ref ( 47 ) ) . in the zebrafish , enzymes 
 structurally and functionally similar to the mammalian adhs have been 
 identified
. however , the affinity of these enzymes to alcohols appears 
 to be less specialized than that for mammalian enzymes but the affinity of each enzyme for the various alcohols has not 
 been studied in detail .
in addition to their role in alcohol metabolism , 
 adhs also convert nad+ to nadh , and this may act as a compensatory 
 mechanism for potential reduction in nadh resulting from the inhibition 
 of the pkm .
the significance of the very pronounced increase in adh5 following exposure to all three forms of ag is , therefore , 
 difficult to ascertain .
we hypothesize that this could be associated 
 with the effects of all treatments on the oxidative phosphorylation 
 pathway that may have resulted in a shift toward anaerobic metabolism , 
 with increased accumulation of alcohol within the cells , and detoxification 
 by adh5 .
analyses for differentially 
 expressed genes across the different treatments and consistent over 
 time ( to investigate for possible particle related effects ) identified 
 1 gene for ag bulk , 13 genes for the agnp treatment and 32 genes for 
 the ag treatment , respectively ( si table s2 ) . these differences
, however , would appear to be relative 
 to the proportion of biologically available ag in the 
 different treatments and may not only indicate unique mechanisms associated 
 with each treatment but also be a function of the level of bioavailable 
 ag . considering the uniquely expressed genes in 
 the different treatments , for ag bulk , ets translocation variant 5 
 ( etv5a ) was 1.4 and 1.5-fold down - regulated at 24 
 h and 48 h , respectively .
this gene is involved in the cellular response 
 to oxidative stress ( go:0034599 ) and in the regulation of transcription , 
 dna - dependent ( go:00063550 ) .
we identified 13 genes that were 
 specific to agnp . among these 
 genes ,
cryptochrome 1a ( cry1a ) was strongly down - regulated 
 both at 24 and 48 h of exposure ( 6.3 and 4.3 fold down - regulated , 
 respectively ) .
this gene acts as a potent repressor of clock function 
 and mimics the effect of constant light to  stop  the 
 circadian oscillator , and is induced by light .
this finding suggests that the light availability to embryos 
 exposed to agnp differed from that available to embryos in other treatments . 
 despite the fact that we tested relatively low concentrations of agnp , 
 it is possible that the suspended nanoparticles , and/or nanoparticles 
 at the surface of the embryo , were acting as a barrier to light , and 

may have resulted in a difference in light availability to the embryos 
 incubated in the agnp treatment .
other genes responsive to agnp alone 
 included the proteasome subunit beta type-1 ( psmb1 ) , a gene involved in protein catabolism ( 1.2- fold down - regulated 
 following 24h of exposure and 1.3-fold up - regulated at 48 h of exposure ) 
 and ribosomal protein s6 modification - like protein b ( zgc:92164 ; 1.7-fold and 1.5- fold up - regulated at 24 h and 48 h , respectively ) . 

together , these changes , although small in magnitude , indicate that 
 the protein turnover in the agnp treatment may be increased compared 
 to the other treatments ( in particular to ag bulk ) , suggesting that 
 the potential damage caused by ros on cellular proteins is compensated 
 through degradation and synthesis of new proteins .
the genes 
 present in this list belong to gene ontologies 
 that overlap with those over - represented among the genes affected 
 by the other treatments ( including oxidative phosphorylation and generation 
 of precursor metabolites and energy ) .
genes regulated included cyclic 
 amp - dependent transcription factor atf-4 ( atf4b1 ; 
 2.3 and 2-fold down - regulated at 24 h and 48 h , respectively ) ; a gene 
 involved in gluconeogenesis and regulation of transcription and probable 
 atp - dependent rna helicase ddx56 ( ddx56 ; 4.3 and 
 4.6-fold down - regulated at 24 h and 48 h , respectively ) , a gene involved 
 in ribosome biogenesis .
this adds to the evidence that the genes unique 
 to this treatment reflect responses to higher levels of biologically 
 available ag associated with this treatment . to 
 summarize ,
we have applied sequencing - based transcription - profiling 
 to investigate the mechanisms of toxicity of agnp , compared to its 
 ionic and bulk counterparts , in exposed zebrafish embryos , and provide 
 mechanistic evidence for the hypothesis that the toxicity of agnp 
 is principally associated with the presence of bioavailable ag .
all ag treatments resulted in an inhibition of the oxidative 
 phosphorylation pathway at 24 h of exposure , potentially due to the 
 increase in ros within mitochondria .
unique gene responses in the 
 different treatments also supported that differences for responses 
 in the transcriptome could be caused by the differences in bioavailability 
 in ag between the treatments .
however , a gene response 
 unique to agnp was identified that may have resulted from alterations 
 in light availability to the developing embryos , demonstrating that 
 not all changes in gene expression observed can be explained by the 
 effects of ag alone , and that particle - associated effects 
 may also contribute to the toxicity of agnp .
our work suggests 
 that the predominant adverse effects of agnp 
 appear to be associated with the toxicity of ag .
we would 
 suggest that the risk assessments for agnp should have a strong focus 
 on understanding the bioavailability of ag originating 
 from agnp , and the toxicology of ag , to establish what 
 the levels of exposure and health consequences are likely to be in 
 vulnerable environmental compartments .
 silver 
 nanoparticles cause toxicity in exposed organisms and are 
 an environmental health concern . the mechanisms of silver nanoparticle 
 toxicity , however , remain unclear . 
 we examined the effects of exposure 
 to silver in nano- , bulk- , and ionic forms on zebrafish embryos ( danio rerio ) using a next generation sequencing approach 
 in an illumina platform ( high - throughput supersage ) . 
 significant alterations 
 in gene expression were found for all treatments and many of the gene 
 pathways affected , most notably those associated with oxidative phosphorylation 
 and protein synthesis , overlapped strongly between the three treatments 
 indicating similar mechanisms of toxicity for the three forms of silver 
 studied . 
 changes in oxidative phosphorylation indicated a down - regulation 
 of this pathway at 24 h of exposure , but with a recovery at 48 h. 
 this finding was consistent with a dose - dependent decrease in oxygen 
 consumption at 24 h , but not at 48 h , following exposure to silver 
 ions . 
 overall , our data provide support for the hypothesis that the 
 toxicity caused by silver nanoparticles is principally associated 
 with bioavailable silver ions in exposed zebrafish embryos . 
 these 
 findings are important in the evaluation of the risk that silver particles 
 may pose to exposed vertebrate organisms . 
Introduction
Materials
and Methods
Results and Discussion