fenitrothion [ o , o - dimethyl o-(4-nitro - m - tolyl)phosphorothioate ] is an organophosphate pesticide widely used as a broad - spectrum contact insecticide for crop protection in the united kingdom , europe , australia , and japan . in the united states ,
fenitrothion is licensed for use in household and industrial pest control products.(4 ) it is classed as a  red list  pollutant by the u.k .
environment agency , that is , one of the most dangerous substances to the aquatic environment whose discharge into water is to be minimized as far as possible,(5 ) and consequently its presence in the environment is of particular concern.(1 ) this pesticide can contaminate surface water from soil runoff,(1 ) with up to 8 g / l reported in streams receiving input from japanese paddy fields,(3 ) up to 150 g / l detected in surface waters in spain,(6 ) and levels in excess of 100 g / l in runoff from an italian vineyard.(2 ) although fenitrothion has been reported to biodegrade relatively rapidly , for example .
4 days in natural waters,(7 ) regular input into the aquatic environment can result in sublethal exposure of nontarget organisms . as for other organophosphates , fenitrothion acts by inhibiting the activity of acetylcholinesterases ( ache ) . while observations of whole - organism toxicity have been documented , including increased mortality rates in bees,(8 ) altered foraging habits in birds,(9 ) and fatigue in mammals,(10 ) no extensive investigations
have been reported into the underlying target and off - target molecular toxicities of fenitrothion .
previous molecular studies have reported that fenitrothion bioaccumulates in the brain , liver , muscle , and blood of exposed fish.(11 ) furthermore , at water concentrations of 40 g / l it can significantly deplete hepatic glycogen and total lipid levels and significantly increase plasma glucose and lactate in fish , with alterations in these biomarkers indicating a shift toward glycolysis - based energy production.(12 ) fenitrothion also reduces 3--hydroxysteroid dehydrogenase activity in carp gonads at exposure levels of 1.5 g / l , with likely impacts on sex steroid metabolism.(13 ) further studies show that fenitrothion acts as an androgen receptor antagonist in vitro , which is associated with reduced prostate and seminal vesicle tissue masses in rats in vivo.(14 ) exposure of stickleback to 10 g / l fenitrothion significantly reduced spiggin ( an androgen - dependent protein used for nest construction ) production,(15 ) while levels of only 1 g / l disrupted their androgen - dependent reproductive behaviors.(16 ) the occurrence of fenitrothion in the environment at concentrations believed to cause sublethal toxicity was a major driver for our investigations into the molecular mechanisms of this pesticide s effects in a fish species .
we chose to study the roach ( rutilus rutilus ) as it is a sentinel species for assessing endocrine disrupting chemicals,(17 ) and is widespread and ecologically important in lowland rivers throughout eurasia . adopting a chronic , environmentally relevant exposure , we aimed primarily to provide a more comprehensive understanding of the molecular toxicity of fenitrothion .
a secondary aim was to identify a potential suite of molecular biomarkers of chronic effect and to discuss their applicability to environmental biomonitoring .
both targeted and nontargeted metabolomic analyses were conducted on selected tissues from male roach following a 28-day exposure to 2 , 20 , and 200 g / l fenitrothion .
metabolomics can be used to investigate metabolic changes within a biological system in response to toxicant exposure or disease , and has considerable potential as a tool in the fields of environmental risk assessment and monitoring.(18 ) for example , nontargeted metabolomic approaches can provide an unbiased determination of metabolic pathway perturbations in response to toxicant exposure and can lead to determination of toxicant mode of action.(19 ) key metabolic changes indentified from such studies , once understood and validated , can then in principle be used to monitor for environmental toxicant exposure . here , targeted metabolite analyses of testes , brain , and plasma were focused on preselected metabolites and/or metabolic pathways that we hypothesized were involved in the toxic response , specifically the disruption of acetylcholine ( ach ) metabolism and steroid metabolism , utilizing liquid chromatographymass spectrometry ( lc - ms ) and immunoassays with high analytical sensitivity and specificity.(20 ) in addition , nontargeted metabolomics of testes and liver was used to simultaneously measure as large a proportion of the metabolomes as possible to investigate for unanticipated metabolic toxicity as well as metabolic degradation products of the pesticide . this utilized both h nuclear magnetic resonance ( nmr ) spectroscopy and direct infusion mass spectrometry ( dims ) -based metabolomics . to further investigate some of the perturbed metabolic pathways identified by the metabolomics approaches , expression levels of selected genes were measured by real - time quantitative polymerase chain reaction ( rt - qpcr ) .
sexually mature roach of a mixed - sex population ( laboratory - maintained first - generation fish derived from wild parental stock ; calverton fish farm , u.k .
environment agency ) were randomly allocated into groups of 12 fish in 150 l tanks .
each tank was exposed to nominal concentrations of 2 , 20 , and 200 g / l fenitrothion ( sigma aldrich ) suspended in ethanol ( each exposure was done in duplicate ; see supporting information for further details ) .
water control ( wc ) and solvent control ( sc , ethanol added ) tanks were run under the same conditions without fenitrothion .
fish underwent a 28-day ( chronic ) exposure under continuous flow - through conditions ( tank water fenitrothion concentrations were confirmed by gas chromatography ( gc)ms ; supporting information ) and then sacrificed by terminal anesthesia with benzocaine .
fork length , wet body mass , condition factor ( fork length / body mass ) , and gonadosomatic index ( gsi ; testes mass / body mass ) were recorded .
the liver , gonads , and brain were dissected rapidly and , along with plasma ( from caudal sinus ) , were frozen in liquid nitrogen and stored at 80 c until analysis .
a section of gonad underwent histology to confirm each fish was male ( see supporting information ) .
metabolites were extracted from brain , testes and liver using a two - step methanol / chloroform / water method with a final solvent ratio of 2:2:1.8.(21 ) extract blanks were produced by the same protocol but in the absence of tissue .
the extracted polar phase was split into 2  50 l aliquots for dims or 1  600 l aliquot for nmr and then dried .
the dims aliquots were resuspended in 80:20 methanol / water containing either 20 mm ammonium acetate ( for negative - ion dims ) or 0.25% formic acid ( positive - ion dims ) .
the nmr aliquot was resuspended in a sodium phosphate - based buffer ( see supporting information ) .
all treatment groups ( wc , sc , and 2 , 20 , and 200 g / l fenitrothion exposures ; see table s1 in supporting information for group sizes ) were analyzed by nontargeted metabolomics .
liver and testes extracts were analyzed by one - dimensional ( 1d ) h and two - dimensional ( 2d ) h j - resolved nmr spectroscopy on a 500 mhz spectrometer equipped with a cryoprobe ( bruker biospin , uk ) .
preprocessing included taking skyline projections of the 2d j - resolved spectra ( termed pjres).(22 ) residual water and chloroform peaks were removed , and spectra were normalized to a total area of unity and then binned prior to statistical analysis , described below(22 ) ( further details in supporting information ) .
liver and testes extracts were analyzed by dims in negative - ion mode by the selected ion monitoring ( sim ) stitching method(23 ) on a hybrid linear ion trap / fourier transform ion cyclotron resonance mass spectrometer ( ltq ft - icr , thermo fisher scientific , germany ) .
each sample was analyzed in triplicate and filtered into a single peak list ( see supporting information ; internal calibrants in table s2 ) .
final matrices containing peak intensity data were normalized by the probabilistic quotient method(24 ) prior to statistical analysis , described below .
selected peaks were subsequently identified by collision induced dissociation - based ms / ms in the linear ion trap ( energy applied 35 ev ) , and fragments were recorded in the ion cyclotron resonance detector .
polar brain extracts ( from sc and 2 and 200 g / l fenitrothion exposures , each n = 6 ) resuspended in methanol / water were analyzed by dims and processed as for nontargeted analysis .
sim windows at 90120 and 130160 m / z were recorded ( positive ion ) to measure choline and ach , respectively .
prior to testes extraction , 2 ng of deuterated testosterone - d3 and 4 ng of deuterated cortisol - d4 were added to each sample ( sample group sizes , table s3 in supporting information ) .
testes were extracted in methanol followed by two solid - phase extractions.(20 ) steroid standards [ cortisone , cortisol , 17-hydroxyprogesterone , androstenedione , 11-ketotestosterone ( 11-kt ) , testosterone , and 11-hydroxyandrostenedione ; steraloids ] were made both with and without a pooled testes extract ( equivalent of 100 mg of testes / sample ) and 2 ng of testosterone - d3 and 4 ng of cortisol - d4 per lc - ms injection . roach samples and steroid standards ( in 20 l of 50:50 methanol / water ) were analyzed by ultraperformance liquid chromatography time - of - flight ms ( waters , u.k.).(20 ) the mobile phases were ( a ) 95% water , 5% acetonitrile ( acn ) , and 0.25% formic acid and ( b ) 100% acn .
the gradient used was 014.9 min , from 20% to 80% b ; 14.915.0 min , from 80% to 100% b ; 15.025.0 min , 100% b. for the analysis of plasma 11-kt , 20 l of plasma was extracted(25 ) and the androgen was quantified by radioimmunoassay.(26 ) total rna was extracted from 10 mg of liver ( from the methanol / water homogenate prepared for the nontargeted metabolomics , n = 8) or 10 mg of testes ( n = 78 ) , by use of the rneasy system with dnase treatment ( qiagen , crawley , u.k . ) according to the manufacturer s protocol ( supporting information ) .
cdna was synthesized from 1 g of total rna by use of random hexamers and moloney murine leukemia virus reverse transcriptase ( promega , southampton , u.k . )
rt - qpcr ( gene - specific primer sequences , table s4 in supporting information ) was performed on relevant target genes including creatine kinase , glutathione s - transferase , phenylalanine hydroxylase , androgen receptor , and gonadal aromatase using the icycler iq real - time detection system ( bio - rad laboratories inc . ) , and expression levels were normalized to expression of the ribosomal protein l8 gene ( further details in supporting information ) .
analysis of variance ( anova ; one - way , with tukey s post hoc testing ) was conducted on whole - organism responses , targeted metabolite levels , and gene expression levels , while the kruskalwallis method ( with dunn s all - pairwise multiple comparison ) was used on data shown to be nonparametric ( 11-kt radioimmunoassay data);(27 ) both methods were conducted in minitab .
to avoid biasing the statistical analyses , peaks in the nmr and mass spectra from fenitrothion metabolism were removed .
specifically , dims peaks corresponding to desmethylfenitrothion [ m  h ] or s - methylglutathione [ m  h ] were removed ( identities confirmed by ms / ms ; figure s1a , b in supporting information ) . in the nmr spectra of liver extracts , bins from 2.83 to 2.86 ppm that were highly suspected to correspond to desmethylfenitrothion ( figure s2 in supporting information ) were removed .
a generalized log transformation was applied to the normalized pjres nmr and dims data sets , and then principal component analysis ( pca ) was conducted using pls_toolbox ( eigenvector research , wenatchee , wa ) in matlab ( version 7 , the mathworks , natick , ma ) .
anova was conducted on the scores for the first five pcs from each model [ with a false discovery rate ( fdr ) of < 10% to correct for multiple hypothesis testing](28 ) to establish significance of treatments .
anova was conducted on each variable ( pjres nmr bin or dims peak ) in the normalized data sets across sc and 2 , 20 , and 200 g / l fenitrothion treatment groups ( at fdr < 10% ) . significantly changing nmr bins
were screened against an in - house nmr library of 210 metabolite standards , and then the definitively identified metabolites were quantified in the 1d biological nmr spectra , normalized to the total spectral area , and tested by anova ( supporting information and table s5 ) .
significant peaks in the dims data were annotated by use of mi - pack software,(29 ) described in supporting information .
metabolite x - fold changes were calculated as the average intensity in a treated group relative to the average of the sc group .
mean ( sem ) measured fenitrothion exposure concentrations were 2.9  0.9 , 30  9.7 , and 193  61
g / l for the nominal 2 , 20 , and 200 g / l exposures , respectively .
no mortalities or significant changes to fork length ( p = 0.543 ) , body mass ( p = 0.439 ) , gsi ( p = 0.542 ) , or condition factor ( p = 0.940 ) occurred during the 28-day experiment ( data not shown ) , confirming that the selected exposure concentrations did not induce overt toxicity .
two peaks were identified by ms / ms as desmethylfenitrothion ( only detected in exposed fish , including within the liver , testes , and brain ) and s - methylglutathione ( 10-fold increase in liver , testes , and brain of exposed fish versus controls , p  3.14  10 ) ( figures s1a , b and s3 and table s6 in supporting information ) .
the normalized intensities of these two peaks in the liver and testes data sets were significantly correlated ( p = 1.7  10 and 2.9  10 , respectively ; figure 1 ) , strongly suggesting that they result from the metabolic breakdown of fenitrothion ( see supporting information for further details ) .
correlations between the ranked intensities of s - methylglutathione [ m  h ] and desmethylfenitrothion [ m  h ] measured in ( a ) liver and ( b ) testes of roach exposed to fenitrothion , confirming that o - demethylation is a major route of pesticide degradation . a peak at 146.117  56 m / z in the dims of brain extract
was confirmed by ms / ms as [ ach - e ] ( 0.035 ppm mass error ; figure s1c in supporting information ) .
[ ach - e ] was depleted following exposure to 2 g / l ( 1.80-fold , significant by tukey s post hoc ) and 200 g / l fenitrothion ( 1.36-fold ) compared to the sc group , confirming disruption at the target site ( anova , p = 0.009 ) .
the intensity of a peak putatively identified as [ choline - e ] ( 104.107  00 m / z ) was not significantly changed ( p = 0.291 ) across the treatment groups .
cortisone showed a significant 4.0-fold depletion , from sc to 200 g / l exposure , in response to fenitrothion ( p = 0.004 , post hoc : 200 g / l group significantly different from wc , sc , and 2 g / l exposure ) , and 11-hydroxyandrostenedione showed a near - significant 2.9-fold increase from sc to 200 g / l exposure ( p = 0.057 , figure 2a , b ) .
standard curves for these steroids showed similar gradients with or without the presence of tissue extract , indicating that the biological matrix exerts minimal ion suppression on them ( figure s4 , supporting information ) .
cortisol ( p = 0.34 ) , androstenedione ( p = 0.87 ) , and 11-kt ( p = 0.93 ) levels in testes extracts did not change significantly on exposure ( figure s5 , supporting information ) , while 17-hydroxyprogesterone and testosterone were below the lc - ms detection limit .
plasma 11-kt levels were significantly ( p = 0.038 ) reduced in response to fenitrothion ( 5.3- and 4.7-fold reductions for the 200 g / l exposure compared with wc and sc , respectively ; figure 2c ) .
mean concentrations ( sem ) of ( a ) cortisone and ( b ) 11-hydroxyandrostenedione in roach testes and ( c ) 11-ketotestosterone in plasma .
key : water control ( wc ) , solvent control ( sc ) , and 2 , 20 , and 200 g / l fenitrothion exposures .
lowercase letters indicate significance , such that bars with the same letter are not significantly different .
pca was conducted on the dims and nmr data sets ( representative spectra , figure 3 ) of testes and liver ( pca scores plots , figure 4 ) , and the resulting scores were evaluated by anova ( table s7 , supporting information ) .
significant differences existed between the five treatments groups ( fdr < 10% ) along two of the top five pc axes for the nmr liver and testes models and for the dims liver model .
no significant differences between treatments were revealed in the pca of the dims testes data set .
pc1 from the nmr liver model and pc5 from the dims liver model showed the highest significance ( p = 8.3  10 and 9.4  10 , respectively ; post hoc : 200 g / l exposure significantly different from all other groups along both axes ) , indicating a greater overall metabolic response in the liver metabolome compared to the testes metabolome ( post hoc summary , table s8 , supporting information ) .
the wc and sc groups were not significantly different ( t test , p > 0.1 in all cases ) along the first five pcs in all models , confirming that the solvent had no significant affect on the observed liver or testes metabolomes .
representative spectra of tissue extracts from an untreated roach : 1d projections of j - resolved h nmr spectra of ( a ) testes and ( b ) liver , and negative - ion direct infusion mass spectra of ( c ) testes and ( d ) liver .
selected metabolites that changed significantly in response to fenitrothion exposure are indicated : ( i ) creatine , ( ii ) phosphocreatine , ( iii ) isoleucine , ( iv ) valine , ( v ) n - acetylphenylalanine , and ( vi ) tyrosine .
pca score plots for ( a ) nmr liver data , ( b ) dims liver data , ( c ) nmr testes data , and ( d ) dims testes data .
key : (  ) water control , (  ) solvent control , and (  , green ) 2 g / l , (  , blue ) 20 g / l , and (  , red ) 200 g / l fenitrothion exposures . the pc axes were selected ( up to pc5 ) based upon which axes showed a significant difference between groups ( fdr < 10% ; see table s7 in supporting information ) , except for panel d , in which no pcs were significant so the axes capturing the highest variance were plotted .
multiple univariate statistical testing across the sc and 2 , 20 , and 200 g / l fenitrothion treatment groups revealed 9.34% and 5.84% of the variables in the nmr and dims liver data sets were significantly different ( fdr < 10% ) , respectively , while 0% and 1.56% of the variables differed significantly in the nmr and dims testes data sets , respectively ( table s7 , supporting information ) .
this indicated that fenitrothion induced a larger metabolic response in the liver versus testes metabolome , in agreement with the findings from the pca .
the nmr liver data set contained 161 significant bins ( of a total of 1724 , fdr < 10% ) and of these , four metabolites ( creatine , phosphocreatine , isoleucine , and valine ) were positively identified ( table s9 , supporting information ) . in the dims liver data set ,
243 peaks ( of a total of 4162 ) were significant ( fdr < 10% ) and of these , 20 were putatively identified from the kegg database ( table s10 , supporting information ) . in the dims testes data set
there were 50 significant peaks ( of a total of 3709 ) , of which 10 were putatively identified from kegg ( table s10 , supporting information ) .
tables s11 and s12 ( supporting information ) provide extensive annotation of the entire 4162-peak liver and 3709-peak testes data sets , comprising m / z data , associated summary statistics ( fdr q - value , metabolite x - fold changes ) , and putative identifications [ empirical formula(s ) and metabolite names from kegg database ] .
table 1 shows a selection of data from tables s9 , s11 , and s12 ( supporting information ) , focusing upon key metabolic changes in the dims and nmr data sets of liver and testes that are discussed below . in the dims liver data set the identities of creatine , phosphocreatine , and tyrosine were confirmed by observation of highly significant correlations between the nmr and dims peak intensities ( table s10 , supporting information ) , which also illustrated the high consistency between the analytical methods for these metabolites ( further highlighted in table s13 in supporting information ) .
complete lists of annotated peaks in the liver and testes data sets are shown in tables s9 , s11 , and s12 in supporting information .
significant changes to creatine and phosphocreatine levels in liver indicated a perturbation to energy metabolism . in the testes , phosphocreatine levels
were substantially increased in the highest exposure compared to sc , though not significantly at fdr < 10% ( table 1 ) . consistent with these findings and strengthening the case for perturbed energy metabolism , a peak putatively identified as the doubly charged ion of atp ( 252.4906
m / z ) showed near - significant changes similar to those of phosphocreatine in testes and liver tissue ( table 1 ) .
significant increases in n - acetylphenylalanine levels ( confirmed by ms / ms ; figure s1d in supporting information ) in both tissues , together with altered tyrosine and phenylalanine levels in the testes and liver dims and liver nmr data sets ( table 1 ) , indicated a fenitrothion - induced effect on phenylalanine metabolism .
relevant target genes were selected partly from prior knowledge of fenitrothion toxicity , for example , androgen receptor ( ar ) and aromatase ( cyp19a1a ) , and partly from the discoveries of perturbed metabolic pathways , for example , glutathione s - transferase ( gst ) , creatine kinase ( ck ) , and phenylalanine hydroxylase ( pah ) . in regard to pesticide degradation ,
testes gst was not significantly changed ( p = 1.0 ) , while hepatic gst expression showed a significant increase in response to fenitrothion ( p = 0.007 ) ; however , wc was not significantly different from any exposed group ( figures s6 and s7 , supporting information ) . concerning energy metabolism , although ck showed no significant change in expression in either liver ( p = 0.20 ) or testes ( p = 0.07 ) , a trend toward decreasing ck expression in the liver with increasing fenitrothion concentration was observed ( figure s6 , supporting information ; 2.0-fold decrease in 200
fenitrothion did not significantly alter pah expression in either testes or liver ( p = 0.05 and 0.64 , respectively ; figures s6 and s7 in supporting information ) .
the measurement of endocrine - related genes in the testes revealed no significant changes in ar ( p = 0.58 ) , while cyp19a1a showed a significant decrease in response to fenitrothion ( p = 0.03 ) ( figure s7 , supporting information ) ; however , sc was not significantly different from , any exposed groups suggesting that the solvent caused this effect .
the metabolic degradation product of fenitrothion , desmethylfenitrothion , was detected in brain , testes , and liver , confirming uptake of the pesticide by roach .
the observed significant increase in s - methyl - gsh in fenitrothion - treated fish , and its significant correlation with desmethylfenitrothion ( figure 1 ) , strongly suggests that both compounds result from metabolic decay of fenitrothion .
organophosphate ( op ) compounds can undergo o - demethylation catalyzed by glutathione s - transferase in the presence of reduced glutathione , producing demethylated op and s - methyl - gsh.(30 ) here we confirm this as a major route of fenitrothion degradation in roach .
the significant depletion of cortisone and suggested increase in 11-hydroxyandrostenedione in the testes , together with the significant depletion of 11-kt in the plasma , confirms that fenitrothion disrupts steroid metabolism in roach .
plasma 11-kt in fish is a much more effective stimulator of secondary sexual characteristics and spermatogenesis than testosterone(31 ) and appears to play a key role in driving male sexual behaviors.(25 ) thus , the effects seen on 11-kt could have a major bearing on sexual function in males .
previously it has been suggested that fenitrothion acts as an androgen receptor antagonist.(14 ) the consequences of such inhibition are 2-fold : first , depending on the extent of inhibition , receptor - mediated androgen signaling will be decreased .
second , over time , androgen synthesis may increase to compensate for this reduction in receptor - mediated signaling.(14 ) androgen synthesis , which occurs in the testes , requires cholesterol as its precursor , as is also the case for glucocorticoid synthesis ( figure 5a ) .
the observed decrease in cortisone and increase in 11-hydroxyandrostenedione in the testes may indicate that steroid synthesis is directed away from glucocorticoids and toward androgens .
since the exposure of roach to fenitrothion was chronic , this may represent an adaptive cellular response to the continued inhibition of the androgen receptor , ultimately enabling androgen receptor - mediated activity even in the presence of the antiandrogenic effects of fenitrothion . the suggested increase in androgen synthesis in the testes was not reflected in the plasma , where 11-kt was significantly depleted .
this may indicate a fenitrothion - induced inhibition of 11-kt secretion from the testes into the plasma , highlighting the importance of studying more than one tissue or biofluid compartment .
overview of metabolic changes discovered in ( a ) steroidogenesis and ( b ) phenylalanine metabolism in roach . observed
metabolites are in boldface type , with arrows ( broad = significant , narrow = near - significant ) indicating the intensity change from solvent control to 200 g / l fenitrothion exposure
. biological compartments of the observations : black arrow = testes ; gray arrow = liver ; open arrow = plasma . data from sancho et al.(12 ) ( i ) phenylalanine n - acetyltransferase ; ( ii ) phenylalanine hydroxylase .
the liver is the major site of op metabolism in mammals , including fenitrothion o - demethylation,(30 ) and fenitrothion has been demonstrated to bioconcentrate in european eel liver.(11 ) the considerable metabolic changes observed in roach liver , which were much greater than in testes , suggest this organ also plays the major responsive role in this species .
energy metabolism was affected in liver and testes , with perturbations to both creatine and the phosphagen phosphocreatine ( table 1 ) ; the latter is known to decrease during high energy demand to buffer atp levels.(32 ) the x - fold change responses of these metabolites differed between tissues , with the liver showing a bimodal dose dependency ( i.e. , metabolites were depleted in the 2 g / l treatment and increased in the 20 and 200 g / l exposures , discussed below ) and testes showing an increase across all fenitrothion doses .
interestingly , it is thought that such a bimodal dose dependency effect is actually more common than the threshold model.(33 ) in mammals , creatine synthesis is initiated in the kidney , completed by the liver,(34 ) and then is distributed to tissues ( including testes ) that have high energy demand .
overall our observations are consistent with the reported disruption to hepatic energy metabolism in eels acutely exposed to 40 g / l fenitrothion.(12 ) another notable and previously undocumented effect of fenitrothion on metabolism , which was consistent across liver and testes , was the increase in n - acetylphenylalanine .
the novelty of this observation is highlighted by the lack of literature on altered n - acetylphenylalanine levels in response to toxicant exposure .
n - acetylphenylalanine is generated from the reaction of phenylalanine and acetyl - coa , catalyzed by phenylalanine n - acetyltransferase .
acetyl - coa can be generated by the breakdown of stored lipids.(35 ) fenitrothion has previously been shown to induce large - scale hepatic lipid depletion,(12 ) which , via acetyl - coa production , could explain the observed significant increase in the acetylation of phenylalanine as well as the observed decreases in both phenylalanine and tyrosine ( figure 5b ) .
the molecular changes detected could potentially act as biomarkers to assess fenitrothion toxicity in field - sampled chronically exposed roach .
our results , however , highlight the considerable challenge of interpreting molecular responses to chronic exposure .
for example , ops primarily exert toxicity via ache inhibition that under acute exposure leads to ach accumulation at nerve synapses .
although the significant effect on brain ach observed here confirms that fenitrothion acts upon this target in roach , the decrease of ach was unexpected and suggests that chronic op exposure results in a degree of adaptation .
this is consistent with a recent hypothesis that ache recovery following chronic op exposure can be overcompensated , leading to increased ache activity and a cholinergic deficit.(36 ) such compensatory shifts in molecular pathways  leading to an adapted phenotype  could also explain the large x - fold increases of phosphocreatine , in liver and testes , in response to the higher fenitrothion doses , again opposite to the anticipated acute response to high energy demand .
metabolic adaptation during the exposure period has been documented previously in a metabolomics study of fathead minnow dosed with 17-ethynylestradiol.(37 ) even if these underlying metabolic changes can be understood , the implications for interpreting molecular biomarker responses in field - sampled organisms are considerable .
? this would be the ideal scenario , providing definitive criteria for field assessment ( but requiring a multi - time - point toxicity study to address this ) .
second , can this phenotype provide sufficient evidence of mode of action(s ) of pollutants and simultaneously reveal downstream ( off - target ) molecular effects that relate to whole - organism health ? our results from this single - toxicant study suggest that the answer is yes but that unanticipated molecular responses should be expected , as adaptation is a complex phenomenon that will be challenging to unravel .
several off - target perturbations were discovered by use of metabolomics , including disruption of phenylalanine metabolism . while this observation is robust , significant , and occurs in both liver and testes  therefore hinting at being a useful biomarker of chronic effect 
it is unproven on two points : first , what are the functional implications of this perturbation ? and second ,
how specific is this response to one pesticide , pesticide class , or even to toxicant exposure in general ?
while the considerable value of metabolomics for the initial discovery of toxicant - induced perturbations to molecular pathways is proven , verifying the value of these discoveries prior to application in biomonitoring is essential and has rarely been achieved to date .
this was addressed recently by hines et al.,(39 ) who confirmed that selected metabolic biomarker signatures ( discovered via metabolomics ) were predictive of physiological toxicity in field - sampled marine mussels .
there are further reasons why shotgun metabolomics approaches will have limited direct application in biomonitoring . by definition ,
hence the threshold for statistical significance for any one response is penalized by the correction for multiple hypothesis testing ( e.g. , using the widely applied false discovery correction method).(28 ) put simply , by such an approach , the primary mode of action of fenitrothion may not have been detected ( significantly ) in the brain if ach was measured as part of a 4000-peak fingerprint .
furthermore , although we have demonstrated the value of metabolomics for detecting changes in both the endometabolome ( roach biochemistry ) and exometabolome ( pesticide metabolism ) , separating these metabolomes in field - sampled fish that have been exposed to chemical mixtures would be a considerable challenge ( at least until automated protocols for large - scale , definitive metabolite identification are developed ) .
overall we regard metabolomics as having an important role in the discovery of molecular pathways that are predictive of both mode of action and whole - organism chronic toxicity , but implementation of this knowledge into environmental biomonitoring should utilize targeted technologies , for example , multiplexed lc - ms.(40 ) arguably a biomarker profile comprising ach , 11-kt , cortisone , creatine , phosphocreatine , and n - acetylphenylalanine could convey considerable diagnostic information on target and off - target chronic toxicity of fenitrothion . in conclusion ,
focusing on the specific actions of fenitrothion , we have confirmed that it significantly disrupts acetylcholine metabolism in roach brain and induces changes in key steroids in testes and plasma .
this confirms that fenitrothion acts both via its primary mode of action and as an endocrine disruptor at environmentally realistic levels . using discovery - driven approaches ,
we have provided the first evidence that fenitrothion can disrupt phosphocreatine regulation , providing a molecular rationale for previous reports of fenitrothion - mediated perturbations to whole - organism energetics .
furthermore , among several off - target effects , we have discovered a perturbation to phenylalanine metabolism that results in a significant elevation of n - acetylphenylalanine in both liver and testes .
 the ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored . 
 fenitrothion , an organophosphate pesticide , is categorized as a  red list  pollutant , being particularly hazardous to aquatic life . 
 it acts primarily as a cholinesterase inhibitor , but evidence suggests it can also act as an androgen receptor antagonist . 
 whole - organism fenitrothion - induced toxicity is well - established , but information regarding target and off - target molecular toxicities is limited . 
 here we study the molecular responses of male roach ( rutilus rutilus ) exposed to fenitrothion , including environmentally realistic concentrations , for 28 days . 
 acetylcholine was assessed in brain ; steroid metabolism was measured in testes and plasma ; and nmr and mass spectrometry - based metabolomics were conducted on testes and liver to discover off - target toxicity . 
 o - demethylation was confirmed as a major route of pesticide degradation . 
 fenitrothion significantly depleted acetylcholine , confirming its primary mode of action , and 11-ketotestosterone in plasma and cortisone in testes , showing disruption of steroid metabolism . 
 metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes . on the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity 
 , we propose that chronic pesticide exposure induces an adapting phenotype in roach , which may have considerable implications for interpreting molecular biomarker responses in field - sampled fish . 
Introduction
Materials and Methods
Results
Discussion