methylmercury ( mehg ) is an environmental contaminant produced from natural or anthropogenic sources of mercury by methylation in widespread sulphate reducing bacteria .
mehg enters the aquatic food chain and accumulates to become a threat for higher - order aquatic mammals and fish , but also to human health through consumption of contaminated fish .
mehg has been shown to be detrimental for human health , with many studies emphasizing its neurological toxicity [ 4 , 5 ] .
the molecular pathway by which mehg exerts its toxicity has been the issue for extensive research .
although mehg seems to induce specific cytotoxic symptoms , one main route for mehg molecular toxicity has yet to be elucidated [ 6 , 7 ] .
however , mehg has a strong affinity for thiol groups , making every cysteine - containing protein a potential target for mehg - binding and disruption , meaning that there may not exist one specific route of toxicity . in the search for a specific molecular mechanism of mehg - cytotoxicity ,
several mechanisms have been suggested for example , oxidative stress [ 9 , 10 ] , excito - toxicological effects , microtubule and cell - structural damage , genotoxic effects , and elevated intracellular ca leading to apoptosis [ 11 , 13 ] .
the occurrence of mehg in seafood has led to a debate regarding health promoting nutrients through fish consumption , versus the risk for contaminant exposure [ 1416 ] .
fish serve as an important source of nutrients , vitamins , and minerals and constitute an important part of a balanced diet .
some of the beneficial nutrients in fish are the long chained marine n-3 fatty acids eicosapentaenoic acid ( epa , 20:5n-3 ) and docosahexaenoic acid ( dha , 22:6n-3 ) , which has shown to be important for optimal cognitive health and neuronal development .
but in addition to its nutritional benefits , fish may also accumulate heavy metals and other environmental contaminants in edible parts , posing an exposure risk for higher - order mammals .
many epidemiological studies have investigated the effects of chronic low - dose fetal exposure of mehg in different geographical locations .
some of these studies report no adverse effects [ 18 , 19 ] , while other studies have reported adverse effects .
.   suggest that dietary effects may be responsible for the discrepancies in mehg toxicity between different geographical localities .
they argue that a study [ 18 , 19 ] , performed at the seychelles which showed no adverse effects , is based on a mainly fish consuming population , while another , performed at the pharoe island   which shows adverse effects , was based on populations consuming mainly whale meat . following this argumentation
, a fish - based diet may contain certain ameliorating nutrients that will reduce the toxicity of mehg .
recently there has been increasing focus on interactions between nutrients and toxicants and how nutrients and the nutrient composition of organisms may affect the toxicity of different environmental contaminants .
reviews have pointed to the lack of research on nutrient - mehg interactions and suggest that an increased focus on nutrient - mehg interaction may increase understanding of mehg toxicological mechanisms .
nutrients can affect mehg toxicity and retention in fish , as shown by bjerregaard et al .   who demonstrated that dietary selenite decreased mehg retention in rainbow trout ( oncorhynchus mykiss ) .
marine n-3 fatty acids , in particular dha , have also been shown to modulate mehg - toxicity in rats , and to possibly modulate mehg neurotoxicity , as demonstrated by in vitro studies .
the aim of this study was to elucidate possible intervening effects of n-3 marine pufa ( dha and epa ) compared to the n-6 fatty acid arachidonic acid ( ara , 20:4n-6 ) on mehg cytotoxicity in atlantic salmon kidney ( ask ) cells .
human embryonic kidney ( hek293 ) cells were included in certain aspects of the study , and mehg - induced toxicity was compared between the two cell types by assessing effects on cell proliferation and death using the xcelligence system .
interaction effects caused by fatty acids on mehg toxicity were screened by investigating known mechanistic effects of mehg , such as uptake of mehg in both cell lines , apoptosis in ask cells , and oxidation of rogfp in hek293 cells .
additionally , we investigated the regulation of transcriptional markers for mehg toxicity and fatty acids metabolism and how dha , epa , and mehg affected these in ask cells .
the cells were grown in leibowitz l15 media with 20% fbs and 1  penicillin / streptavidin / amphotericin ( all from sigma , st .
hek293 cells were a gift from marc niere ( university of bergen , norway ) .
louis , mo , usa ) supplemented with 10% fbs , 1  l - glutamine , and 1  penicillin / streptavidin / amphotericin at 37c with 5% co2 .
fatty acids were coupled to fatty - acid - free bsa ( faf - bsa ) ( paa , pasching , austria ) as described by .
briefly , the fatty acids were weighed and 0.04  ml chloroform per mg fa was added .
furthermore , potassium hydroxide ( koh ) was added in a 1  :  3 relationship and the vial shaken in a whirl mixer for ten minutes .
faf - bsa was added in a 2.5  :  1 ratio to fa .
the solution was then shaken 45 minutes , sterile - filtered , and stored anoxic at 80c until use .
ask cells were seeded in concentrations of 24  000 cells per cm . after 24 hours
the cells were preincubated with 300  m dha , 300  m epa , or 300  m ara , all purchased from sigma , st .
louis , mo , usa . the controls were preincubated with equivalent amounts of bsa as would be found together with fatty acids at concentrations of 300  m .
the cells were preincubated with fatty acids or bsa for 72 hours , and the media were removed before mehg , diluted in media , or control , just fresh media , was added .
hek293 cells were seeded , and similar to ask they were preincubated with the same fatty acids ; however , the incubation time was reduced to 24 hours due to a more rapid cell growth of hek293 compared to ask .
mehg , or control , was added and endpoint measurements were performed within a time frame of 48 hours .
48 hours after mehg addition , the cells were scraped off using a rubber policeman and washed 3 times in 1  pbs .
the cells were further divided into two aliquots , where one was added to a dma 80 mercury analyzer .
the other half of the cells were lyzed , and total protein was measured using the bca protein assay ( thermo scientific pierce , rockford , il , usa ) on a labsystems iems reader ( thermo fisher scientific inc . ,
24 hours after mehg addition , cells were harvested and analyzed as described previously for ask cells . in order to investigate the effect of mehg on cell viability and proliferation
, we implemented the xcelligence rtca sp impedance assay from roche diagnostics ( mannerheim , germany ) where cell adherence is measured in real time .
the base of the xcelligence technology is the disposable e - plates , which are similar to normal 96-well microtiter plates .
these plates are connected to an rtca analyzer and computer with rtca - associated software .
the e - plates contain gold - plated sensor electrodes in the bottom of each well , which enables impedance readings from a small current emitted into the system .
the distance from one electrode to the next in the xcelligence system is possibly obstructed by cell media and cells adhering to the electrodes .
cells will act as insulators at the electrodes and will alter impedance measurement based on cell density and cell adherence .
hence , this impedance measurement will correspond to cell growth , cell toxicity , and adherence qualities ( morphology ) of the cells .
a broader introduction of the xcelligence system and software has previously been presented in [ 27 , 28 ] .
ask cells were seeded at a concentration of 24  000 cells per cm in a 96-well e - plate .
fresh media containing bsa were added 24 hours after seeding , at a concentration of 150  m ( equivalent to the amount incorporated in fa ) .
the cells were incubated another 72 hours , before they were washed twice in fresh media and mehg was added in concentrations from 1 to 4  m mehg dispersed in fresh media .
impedance measurement was recorded every 30 minutes throughout the analysis and up to 48 hours after addition of mehg .
hek293 cells were seeded at a concentration of 15  000 cells per cm in a 96-well e - plate .
the cells were incubated for another 24 hours , before they were washed twice in media , and mehg dispersed in media was added in concentrations of 17  m .
impedance measurement was recorded every 30 minutes , with the exception of the first hour after any modification to the cells , where it was measured every 15 minutes .
impedance measurements were performed throughout the analysis and up to 48 hours after addition of mehg .
after addition of mehg to both ask and hek293 , the xcelligence plot showed a pronounced increase in cell adherence .
since this also occurred in the control , it was deemed likely that this specific effect was due to the washing of the cells with the resulting loss of cell adherence and increase in floating cells .
when these cells adhered during the next couple of hours , a pseudoincrease in growth curve would be expected . moreover ,
the renewal of the media with fresh nutrient was also likely to impose a sudden increase in growth of the cells . in order to focus on the more long - term effects of mehg
, it was chosen to normalize cells when the growth curve stabilized . in order to standardize this normalization , growth curve of control cells in
both ask and hek cells was smoothed using the prism 5.04 ( graphpad software inc . , san diego , ca , usa ) , to get more continuous curves .
after smoothing , the first derivate of the curve was calculated in order to investigate the slope of the curve . at the time point
where the first - derivative first approached zero ( where cell growth stabilized ) , the curve was normalized in all treatments for the respective cell type .
ask cells were seeded at a concentration of 24  000 cells per cm in 24-well culture plates and further treated as described in basic experimental design . after 48-hour exposure to mehg , the cells were fixed with 4% para formaldehyde ( chemi teknik , oslo , norway ) and stained with 4  g ml bisbenzimide h 33342 ( sigma , st .
the fluorescent imaging was performed at the molecular imaging center ( fuge , norwegian research council ) , university of bergen .  
ask cells were preincubated with the different fa and exposed to mehg in 6-well plates at a cell density of 24  000  cells / cm , as previously described .
after exposure to mehg for 48 hours , the cells were harvested using a rubber policeman .
cells were lysed , and total rna was extracted using rneasy columns ( qiagen , oslo , norway ) .
the quantity and quality of the rna were assessed with the nanodrop nd-1000  uv - vis spectrophotometer ( nanodrop technologies , wilmington , de , usa ) .
the concentration of rna in one well of a 6-well plate containing 24  000 cells per cm was approximately 50  ng l .
the expression of 11 target genes and two reference genes in ask cells after fa and mehg exposure ( table 1 ) , was analyzed using a two - step real - time rt - pcr protocol .
the rt reactions were run in triplicates in a 96-well plate , with 250  ng rna in each sample . for pcr efficiency calculations a 6-fold serial dilution ( 1000 - 31  ng ) in triplicates
template controls ( ntc ) and rt enzyme control ( nac ) were included in each 96-well plate .
the rt reaction was performed according to manufacturer 's instructions using taqman reverse transcription reagents ( applied biosystems , foster city , ca , usa ) on a geneamp pcr 9700 machine ( applied biosystems , foster city , ca , usa ) .
reverse transcription was performed at 48c for 60  min by using oligo dt primers ( 2.5  m ) for all genes in 30  l total volume .
the final concentration of the other chemicals in each rt reaction was mgcl2 ( 5.5  mm ) , dntp ( 500  mm of each ) , 10x taqman rt buffer ( 1x ) , rnase inhibitor ( 0.4  u/l ) , and multiscribe reverse transcriptase ( 1.67  u/l ) ( applied biosystems ) . diluted cdna ( 1  :  2 ) ( 2.0  l cdna from each rt reaction ) was transferred to a new 96-well reaction plate and the qpcr run in 10  l reactions on the lightcycler 480 real - time pcr system ( roche applied sciences , basel , switzerland ) .
real - time pcr was performed by using sybr green master mix ( lightcycler 480 sybr green master mix kit , roche applied sciences , ) , which contains faststart dna polymerase and gene - specific primers ( 500  nm ) .
pcr was achieved with a 5  min activation and denaturizing step at 95c , followed by 45 cycles of a 15  s denaturing step at 95c , a 60  s annealing step and a 30  s synthesis step , at 72c .
target gene mean - normalized expression ( mne ) was determined using a normalization factor calculated by the genorm software   based on two selected reference genes , ef1ab and arp .
genorm determines the individual stability of a gene within a pool of genes and calculates the stability according to the similarity of their expression profile by pairwise comparison , using the geometric mean as a normalizing factor .
the gene with the highest m , that is , the least stabile gene , is then excluded in a stepwise fashion until the most stabile genes are determined . here ,
a normalizing factor based on two examined reference genes was used to calculate the mne . in the fluorescent vector pegfp - n , already containing a c48s mutation , serine and glutamine at positions 147 and 204 have been replaced by two cysteines to create the rogfp2 c48s / s147c / q204c .
the thiol group in these cysteines reacts to changes in the red - ox environment of the cell , altering the conformation of the rogfp - protein , and subsequently its conformation and fluorescent properties .
the rogfp has two emission peaks ( figure 1(a ) ) , which vary in intensity according to red - ox status in the cell . by measuring the emission of the rogfp at these two different excitation wavelengths
, a ratiometric value representing the red - ox status in the cell can be obtained .
the plasmid pegfp - n1/rogfp was purchased from university of oregon . a stable cell line expressing rogfp ( figure 1(b ) )
was made by transfecting hek293 using fugene hd transfection reagent ( roche diagnostics , mannheim , germany ) according to manufacturer instructions , and then the cells were selected using 400  g ml geneticin ( g418 ) ( invitrogen , by land ) .
after selection , the cells were grown in media containing 200  g ml geneticin .
cells containing rogfp were excited at 400 and 488  nm , and emission at the two different excitation points was measured at 510  nm using a optima fluostar plate reader ( bmg labtech , offenburg , germany ) .
a ratiometric value from the emission at the two excitation points was obtained by 
 ( 1)emission  at  excitation  point  488  nm  emission  at  excitation  point  405  nm  = red / ox  ratio . 

when rogfp positive cells grow , the total signal will increase , and since the two peaks measured have different emission maxima , a general increase in signal leads to a small change in the ratio . in order to adjust for this every treated sample was normalized to control at each time point : 
 ( 2 )  red  ox  ratioaverage  control  red  ox  ratio  = relative  red / ox  ratio . 
 in order to reduce systematic errors in the assay , the relative red - ox ratios were also normalized to their relative red - ox ratio at the start of the experiment ( t0 ) : 
 ( 3)relative  red  ox  ratiored  ox  ratio  at  t0=normalized  red  ox  ratio . 
 to compare red - ox ratio of fatty acids and mehg to a known oxidative inducer ,
a titration curve using h2o2 was run and a standard curve was calculated using linear curve fitting ( figure 1(c ) ) of the log - transformed h2o2 concentration .
oxidative effects of fatty acids and mehg were then calculated into corresponding concentrations of h2o2 , to visualize oxidative effect .
all statistic analysis was performed using statistica 9 ( statsoft inc . , tulsa , usa ) and graphpad prism 5.04 ( graphpad software inc .
uptake data , apoptosis count , and red - ox measurement were processed using one - way anova followed by post hoc dunnett 's test .
real - time rt - pcr data were processed using the nonparametric kruskal - willis with post hoc paired comparisons .
cells were preincubated with the marine fatty acids : dha or epa , the n-6 polyunsaturated fatty acid ara , or bsa as control , before addition of mehg .
ask cells preincubated with dha , epa , or ara showed no difference in uptake of mehg compared to mehg - control ( figure 2(a ) ) . in hek293 cells , however , dha significantly decreased the uptake of mehg ( figure 2(b ) ) .
cell toxicity of mehg in both ask and hek293 cells was investigated using xcelligence impedance measurement .
both cell lines showed a clear dose - response curve after titration versus mehg ( figure 3 ) . in hek293 ,
impedance measurements were stabilized at a concentration of 5  m  mehg , where no further decrease was measured .
therefore , concentrations above 5  m were considered as too high to be statistically relevant , and only data from 05  m  mehg were used in regression analysis of dose response .
impedance measurement of ask cells also seemed to reach a plateau as the mehg concentration increased similar to hek293 , but more noticeable was the early onset of toxicity from control to 1  m  mehg ( figure 3 ) .
area under curve ( auc ) was calculated in ask cells from time point 102  h144  h after seeding , as a measurement of cell toxicity , and used in regression analysis .
this dose - response curve of ask auc followed a nonlinear regression curve with r = 0.9824 ( figure 3(c ) ) .
auc was also calculated from hek cells from time point 5396  h. the dose - response curve of hek auc followed a linear curve fitting with r = 0.9491 ( figure 3(d ) ) .
apoptosis was measured using morphological analysis of ask cells after fatty acid preincubation and mehg exposure .
a dose - response curve of mehg was investigated by preincubating cells with bsa , instead of fatty acids , followed by a titration to different mehg concentrations .
apoptosis count was plotted , and curve fitting was performed ( figure 4(a ) ) .
the dose response followed a nonlinear regression curve ( r = 0.9564 ) . from the dose response curve we chose a concentration of 2.5  m  mehg to test the effect of fatty acid preincubation on mehg - induced apoptosis , since this particular concentration of mehg induced a level of apoptosis which can be remedied by molecular intervention .
cells preincubated with different fatty acids were compared to control cells preincubated with bsa and exposed to mehg using one - way anova and post hoc dunnett 's test .
preincubating dha significantly ( p = 0.000014 ) increased while epa significantly ( p = 0.043948 ) decreased apoptosis induced by 2.5  m  mehg .
total rna from ask cells , preincubated with the different fatty acids and exposed to mehg , was extracted and the gene expression of selected markers was investigated . when comparing all groups exposed to mehg versus control ( mehg main effect ) , the b - cell lymphoma 2 ( bcl2 ) like protein ( bclx ) , heat shock protein 70 ( hsp70 ) , and cyclooxygenase 2 ( cox2 ) were all upregulated by mehg ( figures 5 and 6 ) . fatty acid transport protein ( fatp ) , carnitine - palmitoyl transferase ( cpt1 ) , and tubulin  ( tuba )
were downregulated by mehg ( figures 5 and 6 ) . when comparing the different fatty acids ( fatty acid main effect ) versus control , no effects were observed .
when comparing all groups , hsp70 was significantly upregulated in ara- and mehg - treated cells compared to control .
fatp was significantly downregulated in cells preincubated with dha compared to the control , while tuba was significantly downregulated in cells preincubated with epa and exposed to mehg compared to the control .
no significant effects of the treatments were observed in glutathione peroxidase 2 ( gpx2 ) or glutathione - s - transferase ( gst ) ( figures 5 and 6 ) .
hek293 cells were stably transfected with rogfp plasmid and showed a clear fluorescence after g418-selection ( figure 1(b ) ) .
these cells were used to investigate effects of fatty acids and mehg on oxidative status in cells .
oxidative stress caused by fatty acids and mehg treatment was compared to equivalent signals generated by the known oxidative inducer h2o2 ( table 2 ) .
incubating hek293 cells with epa and ara did not induce any change in oxidative status however , both dha and mehg significantly induced an oxidative state in the cells after 5 minutes of incubation ( figure 7(a ) ) .
dha steadily kept cells in a state of oxidative stress during its preincubation , but after addition of fresh media a normal oxidative status was recovered ( figure 7(b ) ) .
mehg also rapidly induced oxidation in the cells , but cells recovered their original oxidative status within approximately 1 hour ( figure 7(c ) ) .
preincubating cells with fatty acids did not affect the change in the oxidative status caused by later mehg exposure ( figure 7(a ) ) .
in this study , the mediating effects of the marine n-3 fatty acids dha and epa on mehg toxicity were studied .
epa ameliorated the apoptotic response of ask to mehg , while dha reduced the uptake of mehg into hek293 cells .
these results indicate that the metabolism of marine n-3 fatty acids may ameliorate mehg toxicity at the molecular level and that the nutrient content of a diet may affect the potential negative effect of mehg - contaminated fish .
however , dha also augmented the mehg - induced apoptosis in ask cells , indicating potential potentiating effects of dha on mehg toxicity .
the metabolism of nutrients into applicable energy for the cell is a vital part in maintaining cell survival . in different exploratory studies performed on fish exposed to mehg ,
changes in metabolic genes or proteins have been reported [ 8 , 31 ] . in our study
we found a significant decrease in cpt1 and fatp gene expression as a result of mehg exposure .
cpt1 is responsible for a rate limiting step in mitochondrial -oxidation of fatty acids : the transport of fatty acids into mitochondria for catabolism .
the regulation of fatp is thought to be governed through peroxisome proliferator receptors ( ppars ) , hence being dependent on induction of ppar through ligand - binding .
fatp is also , in part , responsible for the import of fatty acids into mitochondria by interacting with cpt1 .
when transcription of both cpt1 and fatp is decreased in our cell system , this may signify a decrease in the availability of fatty acids as substrate for energy metabolism in the mitochondria and a subsequent reduced metabolic throughput .
this could be a direct effect of mehg on the proteins themselves or their regulatory mechanisms , or alternatively it could be that the transcriptional expression of these genes is downregulated as a secondary response to metabolic shutdown after mehg - induced apoptosis is initiated .
studies have shown that mehg can disrupt the mitochondrial energy metabolism more directly , possibly through inhibiting phosphorylation of adp to atp , or depolarizing the mitochondria .
the detrimental effect of mehg on mitochondrial metabolism in the cell may also increase leakage of reactive oxygen species from mitochondria and thereby increasing ros .
an important metabolic pathway for 20 carbon polyunsaturated fatty acids ( epa and ara ) is the eicosanoid pathway , where activation of cytosolic phospholipase a2 ( cpla2 ) catalyzes the breakdown of membrane phospholipids and releases ara for subsequent metabolite formation .
an important enzyme in this pathway is cyclooxygenase 2 ( cox2 ) , which we showed to be upregulated in response to mehg exposure .
this may be a secondary response to mehg - induced oxidative stress since cox2 expression has been shown to increase in response to oxidative stress [ 40 , 41 ] .
cox2 is responsible for the conversion of ara into prostanoids , such as the prostaglandins .
an increase in production of prostaglandins may increase the inflammatory response in the cell , meaning that mehg may induce proinflammatory metabolites of ara .
metabolites derived from ara such as the prostaglandin e2 have also shown to increase intracellular ca in osteoblast - like cells .
increase of intracellular ca is also a known toxic effect of mehg , meaning that increased cox 2 leading to increased production of prostaglandins may be part of the underlying cause of mehg - induced ca influx , with consequent increase in apoptosis .
the enzymes in the eicosanoid pathway have the ability to utilize both ara and epa as substrate for production of different metabolites .
however , ara- and epa - induced eicosanoids exhibit different molecular effects in the cell .
in our study , epa ameliorated the apoptotic effect of mehg in the ask cells , while ara did not .
if excess epa out competes ara in the eicosanoid pathway , the resulting metabolites show more anti - inflammatory effects than the respective ara metabolites .
epa might even reduce the inflammatory effects of ara - metabolites created in the same system .
the abandoning of aras proinflammatory and possible ca releasing effects could be part of the explanation why epa reduces mehg - induced apoptosis in ask cells .
ara supplementation did not augment the toxicity of mehg , which one might expect if the proinflammatory ara - derived eicosanoids are damaging to the cell . however , the fbs added to the cell media in the mehg - control probably already contains relatively high ara - to - epa ratio , making the production of ara - derived metabolites saturated , and not dependent on cox2 or the availability of substrate .
another side of the story is that by replacing ara with epa in the eicosanoid system , more epa - derived eicosanoids will be produced , which could reduce inflammation and affect mehg toxicity directly , or through apoptosis signaling pathways .
the effect of epa - derived eicosanoids on mehg toxicity generally , and apoptosis specifically , is not yet known , and further investigations are necessary to elucidate this .
oxidative effects of fatty acids with and without mehg were investigated using rogfp - hek293 cells , and as opposed to dha , epa and ara did not affect the oxidation of rogfp in the cells .
these results are consistent with the lack of antioxidant protection against mehg of fish oil , shown in previous studies .
there are , however , studies showing that epa can cause oxidative stress in muscle and liver cells of atlantic salmon [ 48 , 49 ]
. however , since we did not see any indications of this , there could be other molecular mechanisms explaining the antioxidant effects noted elsewhere in the literature .
epa , unlike dha , did not affect the uptake of mehg into hek293 cells , suggesting that there must be intracellular molecular mechanisms responsible for epa 's ameliorating effects on mehg - induced apoptosis .
dha preincubation significantly decreased the uptake of mehg in hek293 cells , which is consistent with other studies using human cerebellar astrocytes and cerebellar neurons .
no significant difference in uptake of mehg due to dha was observed in the ask cells .
however , ask cells were grown at lower temperature and displayed much lower metabolic rate than hek293 cells . given that uptake of mehg is an active metabolic process linked to carrier mediated processes , the effect of dha on mehg uptake may be disguised in our experimental settings .
unfortunately , this leaves our results in ask cells indicative instead of conclusive , and further research of uptake mechanisms affected by dha , in different cell types , is needed .
the amended uptake of mehg observed in the hek293 cells may be due to altered membrane properties after dha preincubation .
pufas are important constituents in cell membranes , where they affect several functions such as membrane organization , elasticity , microdomain formation , and permeability of the lipid bilayer . however , due to the difference in chain length and the number of double bonds , dha is thought to differ in flexibility and conformational freedom compared to epa and hence will cause different structure disorganization . by changing physical properties of cell membranes , dha may affect uptake of extracellular compounds , such as mehg , into cells .
although dha decreased the uptake of mehg in the hek293 cells , we observed an increase in apoptosis of ask cells after dha preincubation and mehg exposure .
this increase was profound , and real - time rt - pcr analysis of the apoptotic regulator bclx in ask cells suggested a similar effect , though not significant compared to cells only exposed to mehg .
the apoptotic effect seems to be triggered by the addition of mehg , since dha treatment alone did not induce increase in apoptosis ( results not shown ) .
a possible explanation for the severe apoptotic effect of dha together with mehg may be due to changes in the red - ox status in the cells . although some literature reports dha as an inhibitor of oxidative stress in certain cells , dha has also been reported to induce oxidative stress and production of ros [ 48 , 55 ] .
mehg is known to be a pro - oxidant , so the observation can be explained by a potential effect of the compounds .
another possible explanation could be that dha has inhibited the cells innate antioxidant defense system over time .
when we investigated oxidation of rogfp in the hek293 cells , both mehg and dha were observed to oxidize rogfp .
dha kept rogfp in a continuous oxidative state , whilst mehg affected the oxidation of rogfp moretemporarily .
epa and ara did not oxidize rogfp in the cells , indicating that the pro - oxidative effect of these fatty acids , compared to dha , has been obliterated or kept under control in the cells .
conversely , no significant effects of dha were noted on the transcriptional regulation of the two antioxidant enzymes gpx2 and gst in ask cells . however , this may be due to normalization of these levels after replenishing control cells with fresh media after the fatty acid preincubation .
in this study we have shown that the marine n-3 fatty acid dha can decrease mehg uptake in hek293 cells as well as increase mehg - induced apoptosis in ask cells .
furthermore , the proapoptotic effect of dha may depend on its ability to induce changes in the red - ox environment in the cell .
to our knowledge , this is the first time that ameliorating effects of the marine fatty acid epa on mehg - induced apoptosis are reported . taken together
, the effects shown by dha and epa on mehg - induced toxicity , in this study , may help explain the differing toxicity observed in response to type of dietary mehg source .
 methylmercury ( mehg ) is a ubiquitous environmental contaminant which bioaccumulates in marine biota . 
 fish constitute an important part of a balanced human diet contributing with health beneficial nutrients but may also contain contaminants such as mehg . 
 interactions between the marine n-3 fatty acids eicosapentaenoic acid ( 20:5n-3 , epa ) and docosahexaenoic acid ( 22:6n-3 , dha ) with mehg - induced toxicity were investigated . 
 different toxic and metabolic responses were studied in atlantic salmon kidney ( ask ) cell line and the mammalian kidney - derived hek293 cell line . 
 both cell lines were preincubated with dha or epa prior to mehg - exposure , and cell toxicity was assessed differently in the cell lines by mehg - uptake in cells ( ask and hek293 ) , proliferation ( hek293 and ask ) , apoptosis ( ask ) , oxidation of the red - ox probe rogfp ( hek293 ) , and regulation of selected toxicological and metabolic transcriptional markers ( ask ) . 
 dha was observed to decrease the uptake of mehg in hek293 , but not in ask cells . 
 dha also increased , while epa decreased , mehg - induced apoptosis in ask . 
 mehg exposure induced changes in selected metabolic and known mehg biomarkers in ask cells . 
 both dha and mehg , but not epa , oxidized rogfp in hek293 cells . in conclusion , marine n-3 fatty acids may ameliorate mehg toxicity , either by decreasing apoptosis ( epa ) or by reducing mehg uptake ( dha ) 
 . however , dha can also augment mehg toxicity by increasing oxidative stress and apoptosis when combined with mehg . 