selenium ( se ) is an essential 
 micronutrient ; levels only 730 
 times greater than required can be toxic . this is a concern in aquatic environments , where anthropogenic activities 
 can release large quantities of se , and include agricultural runoff 
 of irrigation waters in arid regions ; waste 
 rock from coal , phosphate , and uranium mining ; and combustion 
 waste from coal burning power plants . se 
 usually enters the waterways in its inorganic forms of selenate ( se+4 ) 
 or selenite ( se+6 ) , which can be taken up by microbes and primary 
 producers and converted into various organic forms , including the 
 amino acid , selenomethionine ( semet ) . consumers , 
 such as fish and birds , are exposed to se primarily in the diet and 
 semet has been shown to be the major form of se in the fish diet . semet has been demonstrated to move through the food chain 
 by trophic transfer to higher - level organisms . following 
 a se poisoning event at belews lake , nc , lemly found se to have bioaccumulated 
 from 519 times in periphyton to 3975 times in the visceral tissues 
 of fish . this is of particular concern 
 for oviparous carnivores , for which maternal offloading may impair 
 development or reproductive success through respective teratogenesis 
 or embryo lethality . because of 
 the potential for biomagnification , traditional water 
 quality measurements of se concentrations may be ineffective . , even these measurements may not provide an accurate picture 
 of se effects on an ecosystem , because fish encounter multiple stressors 
 in their environments , which can alter se toxicity . this is of particular importance in areas such as the san 
 joaquin river valley , ca , and the san francisco bay delta area , where 
 many historically freshwater these areas are often spawning grounds for protected 
 species such as the endangered delta smelt ( hypomesus transpacificus ) and threatened steelhead trout ( oncorhynchus mykiss ) . several studies point to oxidative stress as one mode of 
 action for se toxicity . however , oxidative stress is most likely only one 
 factor influencing semet toxicity . the unfolded protein response ( upr ) 
 is a cellular and molecular response to perturbations in endoplasmic 
 reticulum ( er ) homeostasis ( see hetz ( 2012 ) for review ) . oxidative stress , calcium disruption , and 
 glycosylation inhibition , can all disrupt protein folding , leading 
 to the accumulation of unfolded proteins in the er . protein folding 
 chaperones , such as bip ( immunoglobulin- binding protein ; grp78 ) , 
 initiate the upr through dissociation from the mediators of the three 
 branches , perk ( protein kinase rna ( pkr)-like er kinase ) , ire1a ( inositol- 
 requiring protein-1 ) and atf6 ( activating transcription factor 6 ) . while the three branches of the response are highly interconnected , 
 they can be generally divided into three categories . the perk branch 
 is responsible for translational attenuation through activating transcription 
 factor 4 ( atf4 ) . the ire1a branch is responsible for transcription 
 of er - associated degradation ( erad ) genes through x box protein 1 
 ( xbp1 ) . and finally , atf6 is responsible for transcription of protein 
 folding enzymes and chaperones . if the response is unable to attenuate 
 the stress , the upr will initiate cell death , often in the form of 
 programmed cell death ( apoptosis ) . we have previously demonstrated 
 that 50 m of semet and hypersalinity 
 treatment for 24 h significantly decreased embryo hatch , decreased 
 total reduced glutathione , and increased flavin containing - monooxygenase 
 ( fmo ) activity in medaka embryos . exposure 
 of japanese medaka embryos to semet under varied salinities did not 
 impact overall se accumulation but significant differences in toxicity 
 were observed . the purpose of the current 
 study was to further elucidate the mechanisms behind semet and hypersaline 
 induced embryo mortality after 12 h of semet treatment at developmental 
 stages not previously examined . we hypothesized that semet would induce 
 oxidative stress , the upr and apoptosis in japanese medaka embryos 
 and that hypersaline conditions would potentiate these effects . this 
 research will aid in the development for site specific monitoring 
 for se in ca . seleno - l - methionine 
 ( purity 98% ) , 1-butanol , phosphoric acid , thiobarbituric 
 acid and all other reagents were purchased from sigma - aldrich ( st . louis , mo ) . a milli - q water purification system ( millipore , billerica , 
 ma ) was used to obtain deionized water . japanese 
 medaka were cultured at the university of california- riverside and 
 housed in a 2:3 ratio of males to females in medium - hard water at 
 27 c and a photoperiod of 14 h light and 10 h dark . viable embryos were determined 
 based on oil droplet migration to the vegetal pole as outlined by 
 kirchen and west ( 1976 ) . nonviable embryos 
 were discarded and viable embryos were placed into 60  15 mm 
 petri dishes containing either freshwater or a makeup of saltwater 
 from the san joaquin river valley ( 2030 embryos per replicate , 
 and 510 replicates per group ) . although , water - borne semet 
 exposures do not represent the most likely environmental exposure 
 ( the primary exposures for semet are dietary or via maternal transfer ) , 
 they are sufficient to study the mechanistic effects of semet on medaka 
 embryos . san joaquin river valley saltwater was prepared in the lab 
 according to a recipe from westlands water district located about 
 10 km south of mendota in the san joaquin river drainage basin , ca . salinity was measured with a conductivity meter , 
 and corresponds to about 13 and 15.3 g / l suspended solids . following 12 h of equilibration in fresh or salt water , the replicates 
 were divided into three groups . the first subset of embryos were frozen 
 80 c to represent a time zero control ( 12hpf ) . other 
 embryos were treated with a 50 m solution of seleno - l - methionine ( cat . s3132 , sigma - aldrich ) in freshwater or saltwater 
 and exposed for 12 h , then collected and frozen at 80 c 
 for analysis ( 24hpf ) . semet concentrations were chosen based on previous 
 research and were intended to represent the upper levels of bioaccumulation 
 measured in embryos . previous studies 
 have also demonstrated uptake of semet into the embryo , indicating 
 this system was an effective exposure method . the final subset of embryos were left in freshwater or saltwater 
 for 24 h to compare to the semet treated 12hpf samples . the medaka embryo - larval 
 toxicity assay was adapted from farwell 
 et al . embryos were collected 
 and treated as above with one replicate equal to 1520 embryos 
 per dish . following 12 h of semet treatment , embryos were rinsed and 
 transferred to new dishes containing freshwater or saltwater . water 
 was changed every other day and dishes were monitored for mortality 
 with removal of dead embryos . embryos were assessed for deformities 
 and terminated immediately . percent hatch and percent of hatched embryos 
 with deformities were recorded . total 
 mrna was isolated from embryos using the lipid tissue rneasy kit ( qiagen , 
 valencia , ca ) following the manufacturers instructions . mrna quantity 
 and quality was measured using the nd-1000 ( nanodrop , wilmington , 
 de ) . mrna ( 1 g ) was converted to cdna using the reverse transcription 
 system ( promega corporation , madison , wi ) , according to the manufacturers 
 instructions . primers were designed using idtdna primerquest 
 software and optimized using pcr miner ( table 1 ) . as no bax gene for japanese medaka 
 has been annotated in the ncbi database , blast was used on the medaka 
 genome ( http://compbio.dfci.harvard.edu/cgi-bin/tgi/blast/index.cgi ) against bax from zebrafish ( danio rerio ) to develop 
 primers . qpcr was performed with the iscript one - step rt - pcr kit with 
 sybr green from bio - rad ( hercules , ca ) , omitting the reverse transcriptase , 
 on a myiq5 thermo cycler ( biorad ) . the samples were denatured and 
 the polymerase activated at 95 c for 5 min , then 40 cycles of 
 10s at 95 c and 30s of 55 c . samples were subject to melting 
 curve analysis from 65 to 85 c in 0.5 c increments with 
 continuous fluorescence measurement . qpcr was analyzed according to 
 schmittgen and livak and fold change 
 was calculated against the 12hpf freshwater controls . all data was 
 compared against the 12hpf controls in order represent how the gene 
 expression changed over the 12h time period , and how the treatments 
 affected this change . rather than observing a discrete point in development , 
 we feel it is necessary to understand how these treatments altered 
 normal development . thiobarbituric 
 reactive substances ( tbars ) were measured to estimate malondialdehyde 
 ( mda ) formation in medaka embryos . embryos 
 ( 1520 ) were weighed and homogenized in 1.15% kcl then centrifuged 
 at 3000 rpm for 5 min at 4 c . samples were run in duplicate on a wallac victor multilabel plate reader ( perkinelmer , waltham , 
 ma ) with excitation at 535 nm and emission at 585 nm . statistical 
 significance was assessed using a student s t - test or 2-way 
 anova in the statistical program r. statistical significance was determined 
 at p  0.05 , unless otherwise noted . if overall 
 significance was determined following two - way anova , tukey s 
 hsd test was performed posthoc . any non - normal data was log transformed . 
 for data that remained non - normal following log transformation , kruskal while 
 there was no difference in hatch between saltwater and freshwater 
 control embryos , 50 m semet significantly decreased embryo 
 hatch in saltwater treated embryos to 3.7% ( figure 1a ) . the median day to hatch was not significantly affected 
 by semet treatment in freshwater or saltwater ( figure 1b ) . semet and hypersaline treatment also significantly increased 
 the deformities in treated embryos ( figure 1c ) . the most common deformities observed were kyphosis , lordosis , 
 craniofacial abnormalities , and yolk sac edema ( figure 1d ) . effects of combined exposure of semet ( 50 m ) and hypersaline 
 conditions on the development and hatchability of japanese medaka 
 embryos after exposure at 12 hpf . ( a ) percent hatch , ( b ) median 
 day to hatch , ( c ) percent deformities in hatched embryos , ( d ) examples 
 of deformities ; control is top image , bottom two images demonstrate 
 lordosis , kyphosis , cranio - facial abnormalities , and yolk - sac edema . each value represents the mean  standard error ( se ) of 510 
 replicates . statistical significance is indicated by differing letters 
 ( two - way anova , tukey hsd test , or kruskal  wallis , dunn s 
 test p  0.05 ) . there 
 was no significant difference in amount of lipid peroxidation between 
 any of the treatments and no difference between embryos at 12hpf and 
 24hpf ( figure 2 ) . bax transcript levels decreased 
 significantly from 12hpf to 24hpf ( 0.2 fold , p = 
 0.011 ) , while caspase 3a levels remained constant ( figure 3a ) . there was no significant difference in bax or 
 casp3a gene expression following treatment with hypersalinity or semet 
 ( figure 3b and c ) . effects of combined exposure 
 of semet ( 50 m ) and hypersaline 
 conditions on lipid peroxidation in japanese medaka embryos after 
 12 and 24 hpf . ( a ) tbars was measured as nmol / g wet weight tissue 
 in freshwater controls at 12 hpf and 24 hpf . ( b ) tbars measured in 
 embryos in freshwater , saltwater , semet in freshwater and semet in 
 saltwater at 24hpf . hydrogen peroxide ( 3% for 3 h ) was run as a positive 
 control . each value represents the mean  se of 510 replicates 
 statistical significance is denoted by differing letters at p  0.05 ( one - way anova , tukey hsd test ) . effects of combined exposure of semet ( 50 m ) and 
 hypersaline 
 conditions on bax and casp3a transcripts in japanese medaka embryos 
 after 12 and 24 hpf . expression of ( b ) bax and ( c ) casp3a in freshwater , 
 saltwater , semet in freshwater and semet in saltwater . statistical significance is indicated 
 by differing letters ( two - way anova , tukey hsd test p  0.05 ) . atf6 gene expression decreased significantly from 12hpf to 24 hpf 
 ( 0.3 fold , p = 0.019 ) , while bip and atf4 expression 
 increased significantly ( 2 fold , p = 0.048 , and 4 
 fold , p = 0.008 , respectively ) . bip mrna was increased 
 in both saltwater and freshwater semet treatments up to 39 fold over 
 12 h controls and 107 fold over 12 h controls in the semet freshwater 
 and semet saltwater treatments , respectively . though there was a trend 
 toward an increase in atf6 expresssion with semet treatment ( p = 0.119 ) , semet and hypersalinity similarly , trends in atf4 expression indicated 
 a potential difference between freshwater and saltwater treatment 
 ( p = 0.07 ) , with semet decreasing atf4 in freshwater , 
 yet increasing it in saltwater . the mechanism of action of semet toxicity is not well understood , 
 particularly in the presence of multiple stressors , which may confound 
 regulatory monitoring . we observed a decrease in hatch following treatment 
 with hypersalinity and semet , while surviving embryos had deformities . previous work at this developmental stage and semet concentration 
 reported a significant decrease in embryo hatch following 24 h of 
 50 m semet treatment in freshwater . while we did not observe this trend , our results were expected considering 
 the duration of semet treatment was half as that previously studied . overall , our results are consistent with a plethora of other data 
 reporting semet s lethal and teratogenic effects in the field , 
 for example , ref ( 10 ) . japanese medaka are a euryhaline species ; adults are able 
 to spawn 
 and embryos hatch in full seawater . we 
 observed no significant difference in toxicity between freshwater 
 and hypersaline controls , indicating that our results are not due 
 to osmotic stress alone . the mechanism 
 behind this remains to be elucidated , however , fmo may play a role . fmos have been shown to oxygenate semet , which may contribute to its 
 toxicity . this increased fmo activity may increase 
 semet oxygenation , which in turn may increase its embryo toxicity . in contrast , studies have shown that semet activation can occur 
 following methioninase generation of methylselenol , which can subsequently generate oxidative stress in rainbow 
 trout embryos . methylselenol has also 
 been implicated in induction of caspase - mediated apoptosis in cancer 
 cell lines . however , considering that 
 neither oxidative stress nor apoptosis was induced in semet and hypersaline 
 treated embryos and that fmo has been found to be induced by hypersosmotic 
 conditions , we conclude that fmo activation may be a major contributor 
 to the observed toxicity . of interest is that semet and hypersalinity 
 did not generate oxidative 
 stress as measured by lipid peroxidation after 12 h of treatment . however , several groups , including ours , have identified oxidative 
 stress as one of the main modes of action of semet toxicity . as lipid peroxidation is an end point for severe oxidative stress , 
 tbars is not sensitive to small changes in cellular redox . furthermore , in this study , tbars was measured 
 in whole embryos and did not consider localized effects . thus , oxidative 
 stress may still be occurring in semet and hypersaline treatments , 
 yet it may not be detected by our assays or was not as high as that 
 observed after 24hpf . while our results 
 do not eliminate oxidative stress as a mechanism of semet induced 
 embryotoxicty , they indicate that other processes may play an important 
 role particularly at the 12 hour time point of exposure . while we 
 observed no difference in whole - embryo lipid peroxidation between 
 12hpf and 24hpf , many studies demonstrate that the redox status of 
 embryos also undergoes great changes throughout development . two contradicting 
 reports demonstrate the changes in redox status of medaka embryos 
 throughout development . wu et al . measured 
 changes in oxidative stress in the whole embryo each day post fertilization 
 using dichloro - dihydro - fluorescein diacetate ( dchfda ; a dye that fluoresces 
 following oxidation ) and found overall reactive oxygen species ( ros ) 
 increased gradually until hatch . in contrast , another group studying 
 medaka development and silver nanoparticle toxicity , found total ros 
 decreased throughout development . however , 
 in addition to total ros , both of these studies examined multiple 
 biomarkers for oxidative stress and found no common patterns between 
 them . hence , the current studies on redox status throughout medaka 
 development confirm that further studies are necessary in order to 
 understand this complex process . another adverse outcome pathway 
 that may contribute to semet toxicity 
 in medaka at this early life stage is the upr . methylselenic acid 
 ( msa ) induced the upr in pc-3 cells , a human prostate cancer cell 
 line and we observed increases in bip 
 expression following semet treatment , suggesting that semet may be 
 disrupting er homeostasis . although not significant , bip mrna expression 
 was higher in semet and hypersaline treated embryos than in embryos 
 in semet and freshwater . this would suggest that a greater upr is 
 being induced in semet and hypersaline conditions , a mechanism that 
 requires further exploration . the trend toward alterations in atf4 
 and atf6 gene expression also indicated a possible role for the perk 
 and atf6 branches of the upr in semet toxicity . perk also activates 
 nrf2 , a major transcription factor for antioxidant 
 genes during oxidative stress . it must be noted that 
 each branch of the upr has a different activation step ( e.g. , atf6 
 splicing , perk phosphorylation ) according 
 to varying time scales and specific types of er stress . ( 2006 ) compared upr responses 
 generated by dithriothreitol ( dtt ; disrupts disulfide bond formation ) , 
 thapsigargin ( tg , inhibits er calcium - dependent atpase ) , and tunicamycin 
 ( tm , inhibits protein glycosylation ) . the atf6 response to dtt was 
 the most rapid of the three branches , yet it was significantly less 
 sensitive to tg and tm . these 
 responses occurred on different time scales and at different magnitudes ; 
 overall , activation of the branches in response to dtt was faster 
 and stronger than to tm and tg . the varied 
 time scales of each branch of the response to different types of er 
 stress indicate the necessity to document the upr throughout development 
 following semet and hypersaline treatment . furthermore , as mentioned 
 above concerning tbars experiments , these gene expression studies 
 can not discern between localized effects . changes in transcription 
 in a few key cells may not result in a distinct difference in fold 
 change in qpcr . the ire1a - xbp1 pathway has been shown to regulate 
 a variety of developmental processes , including plasma cell differentiation , liver development , chondrogenesis and adipogenesis . the perk / atf4 branch of the upr has been found 
 to be involved in osteoblast formation . bip also plays a major role in development as bip knockout mice 
 are not viable after the peri - implantation stage . the importance 
 of these responses has been found to be conserved in the medaka . recent work established that atf6a / b results 
 in embryonic lethality in medaka , as in mice . they found the physiological response to be strong at 2 days post 
 fertilization , where it was localized to the brain , otic vesicle and 
 notochord . perturbations in the physiological 
 er stress response during embryogenesis may result in the inability 
 of the embryo to manage the high demand for protein folding . if the 
 perturbation is not stopped it could result in teratogenesis and embryo 
 lethality . lordosis , kyphosis , and craniofacial abnormalities were 
 the primary forms of teratogenesis documented in this study . considering 
 the studies above illustrating the role of the upr in the development 
 of the notochord and cartilage , it is possible that upr disruption 
 may be generating the deformities witnessed here . we had hypothesized that apoptosis played a 
 significant role leading to embryo lethality generated by semet . wu et al . demonstrated that 5 m msa was able to 
 cause apoptosis following upr induction in pc-3 cells . however , msa 
 is highly redox reactive and not a natural form of se in the fish 
 diet . bip has been shown to be a negative regulator of apoptosis , so the high levels of bip mrna induction observed 
 may indicate a repression of apoptosis . autophagy is another well - documented 
 outcome from upr , and may also contribute 
 to the deformities and reduction of hatch observed . the comparison 
 of apoptosis taken at 12hpf and 24hpf clearly shows 
 that these important processes fluctuate greatly during development . indeed , apoptosis has been shown to fluctuate in tissue - specific patterns 
 throughout japanese medaka development , occurring mostly in the head , 
 spinal column , and tailbud . the overwhelming evidence for changes in apoptosis , 
 oxidative stress , 
 and the upr throughout development , indicates that developing japanese 
 medaka embryos have important windows of susceptibility to semet and 
 hypersaline stress . thus , while the roles of oxidative stress and 
 apoptosis in the developmental toxicity of semet may be limited from 
 12 to 24 h , they may be increased as the oxidation state of the embryos 
 increases , or as apoptosis is more active at later stages of development . it is important to map these processes throughout embryogenesis , so 
 that we may better understand the developmental toxicity of semet 
 and hypersaline conditions . in summary , hypersaline conditions 
 derived from the san joaquin 
 river valley , ca , enhanced the toxicity of semet in the developing 
 medaka embryo . while the upr may have played a role , oxidative stress 
 and apoptosis measured in the whole embryo additional 
 studies will further consider the role of oxidative stress and the 
 upr throughout medaka development and investigate developmental periods 
 most susceptible to semet and hypersaline toxicity . effects of combined exposure 
 of semet ( 50 m ) and hypersaline 
 conditions on bip , atf6 , and atf4 transcripts in japanese medaka embryos 
 after 12 and 24 hpf . ( a ) expression of bip , atf6 , and atf4 in whole 
 embryos at 12hpf and 24hpf in freshwater . expression of ( b ) bip , ( c ) 
 atf6 , and ( d ) atf4 in 24hpf control embryos and semet treated embryos 
 in freshwater and saltwater . statistical significance is indicated by differing letters ( two - way 
 anova , tukey hsd test p  0.05 ) .
selenium 
 ( se ) is an essential micronutrient that can cause embryotoxicty 
 at levels 730 times above essential concentrations . 
 exposure 
 to hypersaline conditions and 50 m selenomethionine ( semet ) 
 decreased embryo hatch and depleted glutathione in japanese medaka 
 embryos without affecting se accumulation . to better understand the 
 impacts of nonchemical stressors on developmental toxicity of se in 
 fish , several adverse outcome pathways were evaluated in the japanese 
 medaka ( oryzias latipes ) . 
 we treated medaka embryos 
 at 12 h post fertilization with 50 m semet for 12 hours in 
 freshwater or in 13 ppth hypersalinity and evaluated the contributions 
 of oxidative stress , the unfolded protein response and apoptosis to 
 reduced hatch . 
 exposure to semet and hypersalinity decreased embryo 
 hatch to 3.7%  1.95 , and induced teratogenesis in 100%  
 0 of hatched embryos . 
 in contrast , treatments of freshwater , saltwater , 
 and semet in freshwater resulted in 89.8%  3.9186.7% 
  3.87 hatch , and no significant increase in deformities . 
 we 
 found no significant differences in lipid peroxidation , indicating 
 that oxidative stress may not be responsible for the observed toxicity 
 in embryos at this time point ( 24 h ) . 
 although significant changes 
 in apoptosis were not observed , we witnessed up to 100 fold increases 
 in transcripts of the endoplasmic reticulum ( er ) chaperone , immunoglobulin 
 binding protein ( bip ) and trends toward increasing downstream signals , 
 activating transcription factor 4 ( atf4 ) and atf6 indicating potential 
 contributions of the unfolded protein response to the effects of semet 
 and hypersaline conditions . 
 these data indicate that multiple adverse 
 outcome pathways may be responsible for the developmental toxicity 
 of se and salinity , and these pathways may be time dependent .