genetic and genome 
 analyses demonstrate that neurodevelopmental 
 and neurodegenerative disorders are multifactorial in nature and include 
 gene  environment interactions
. these studies 
 define specific molecular mechanisms and cellular pathways that , when 
 perturbed by multiple gene variants , contribute to neuronal disease 
 or dysfunction .
one such essential molecular pathway involves protein 
 ubiquitination , which regulates protein trafficking and activity within 
 the cell and protein degradation through the ubiquitin proteasome 
 system ( ups ) .
protein ubiquitination is particularly important 
 in regulating key activities in the brain , including cellular proliferation , 
 synapse development , neuronal transmission , and synaptic plasticity .
genetic mutations and gene variants of proteins in the ubiquitin 
 cascade have been identified as contributing to multifactorial disorders 
 such as parkinson s disease ( pd ) , suggesting that the nervous 
 system is susceptible to impaired or altered function of protein ubiquitination .
e1 catalyzes the initial 
 atp - dependent thioester formation of ubiquitin to e1 ( ub - e1 ) required 
 for passing ubiquitin on to an e2 conjugase prior to final substrate 
 ubiquitination by either an e2 conjugase or an e3 ligase .
thus , it 
 stands to reason that perturbation of e1 function either through aberrant 
 cellular processes or effects of environmental factors may disrupt 
 a multitude of downstream events dependent upon protein ubiquitination 
 and potentially contribute to the pathogenesis of nervous system disorders .
epidemiological studies support environmental toxin exposure 
 as 
 a risk factor for neurodevelopmental and neurodegenerative diseases .
one recent report suggests that exposure to certain 
 pesticides , including some dithiocarbamates , increases the risk for 
 pd and that specific genetic variants in genes associated with the 
 ups can potentiate this risk significantly .
furthermore , animal and cell culture models demonstrate that exposure 
 to environmental toxins can perturb ubiquitin - based protein processing 
 and can be associated with aberrant neurological outcome .
importantly , specific deleterious effects of pesticide exposure 
 on activation of e1 in vitro and in vivo have been reported .
for example , 
 cultured ventral mesencephalic neurons exposed to ziram , a dithiocarbamate 
 widely used as a fungicide , produced inhibition of e1 , loss of tyrosine 
 hydroxylase positive neurons , and elevated -synuclein levels . mice
administered ziram demonstrated loss of 
 tyrosine hydroxylase positive neurons in the striatum and motor deficits , 
 suggesting that similar effects on e1 may be occurring in vivo .
thus , 
 e1 presents a putative molecular target through which environmental 
 factors can contribute to neurodevelopmental and neurodegenerative 
 diseases .
one potential mechanism of ziram - mediated inhibition 
 of e1 is covalent 
 adduction , a previous study identified e1 as a susceptible target 
 for adduction through both michael addition and sn2 mechanisms 
 in hek293 cell extracts .
e1 was specifically 
 adducted on cysteine 234 , a cysteine located in the first catalytic 
 cysteine domain ( fcch ) of the ubiquitin - binding groove .
subsequent 
 in vivo proteomic studies of rats exposed to n , n - diethlyldithiocarbamate ( dedc ) identified s - ethylaminocarbonyl adducts on brain e1 at cys234 and cys179 accompanied 
 by inhibition of e1 .
furthermore , rats exposed to dedc exhibited markers 
 of neurodegenerative change within the nigrostriatal system similar 
 to those present in pd . while those 
 previous proteomic studies are consistent with inhibition 
 of e1 through electrophilic adduction , other studies report that e1 
 activity is dependent on the redox status of the cell , suggesting 
 that dithiocarbamates may also inhibit e1 through elevated oxidative 
 stress .
dithiocarbamates form metal complexes that 
 vary in their solubility and redox activity depending upon the polarity 
 of the nitrogen substituents and the redox potential of the metal , 
 respectively .
dithiocarbamates readily bind 
 copper and dithiocarbamates with nonpolar nitrogen substituents , including 
 dedc and n , n - dimethyldithiocarbamate ( dmdc ) , form 
 redox active copper complexes that cross cell membranes , partition 
 into lipid compartments , and promote oxidative injury .
this property has been demonstrated in animal models through elevations 
 of copper , lipid peroxidation , protein oxidative injury , and increased 
 apoptosis . in the study presented here , we investigated the 
 relative contribution of covalent adduction and oxidative stress in 
 the dithiocarbamate - mediated inhibition of e1 in hek293 cells .
hek293 cells were obtained 
 from american type culture collection ( atcc ) bioresource ( manassas , 
 va ) and cultured at 37 c in 5% co2 in eagle s 
 minimum essential media ( emem ) ( atcc , manassas , va ) supplemented with 
 10% fetal calf serum , 100 g / ml gentamycin , and 2 mm l - glutamine ( gemini bioproducts , sacramento , ca ) in 75 cm culture flasks until they were 7080% confluent .
cells were 
 washed once with phosphate buffered saline ( pbs ) , incubated at 37 
 c in 1 ml of accutase ( millipore , temecula , ca ) to detach the 
 cells from the flask surface , and resuspended in 10 ml of media , and 
 the cell concentration was determined with a scepter 2.0 automated 
 cell counter ( millipore , temecula , ca ) .
cells were plated at 1.0  
 10 cells / plate in 100 mm  20 mm cell culture plates 
 ( thermoscientific , waltham , ma ) and incubated as described above until 
 they were 7080% confluent .
cultures were supplemented with 
 media as required , and fresh media was added 12 h prior to experimental 
 treatment .
exposure of cells to test compounds was performed as follows : 
 compounds were dissolved in dmso or distilled water , depending on 
 solubility , and a 1/1000 dilution of each was added to emem supplemented 
 as described above .
the main compounds utilized in the study were 
 10 m ziram ( dissolved in dmso ) ( sigma - aldrich , st .
louis , mo ) , 
 20 m dimethyldithiocarbamate ( dmdc ) ( dissolved in dmso ) ( thermo - fisher 
 scientific , waltham ma ) , and either 60 or 120 m triethylenetetramine 
 hydrochloride ( tet ) ( dissolved in water ) ( sigma - aldrich , st .
all controls contained the equivalent amount of dmso ( 0.1% ) as 
 experimental cultures , and preliminary experiments were done to confirm 
 that cell viability was unaffected at this concentration .
other compounds 
 utilized are summarized in table 1 , including 
 information on acquisition of the compound .
culture plates were obtained 
 from the incubator , spent media was aspirated , the cells were washed 
 once with 5 ml of pbs , and 8 ml of fresh media containing the designated 
 compound or control was added to the cultures .
plates were returned 
 to the incubator for 6 h and then processed for sample collection .
hek293 cells were incubated in the 
 presence of the above compounds for 6 h as described in the materials and methods .
the mean ratio of ub - e1/e1 
 values ( + se ) for each treatment group was calculated and compared 
 to control , using one - way anova and tukey s multiple comparison 
 test ( * , p < 0.05 ; * * , p < 
 0.01 ; * * * , p < 0.0001 ; n = 5 
 for all groups ) .
the level 
 of ub - e1 inhibition is 
 calculated as follows : ( od ub - e1/e1compound)/(od ube1/e1control ) .
pydtc , pyrrolidine dithiocarbamate ; zndedc , zinc 
 diethlydithiocarbamate ; nadedc , sodium diethyldithiocarbamate ; eptc , s - ethyl dipropylthiocarbamate ; sadc , sarcosine dithiocarbamate ; 
 dact , diaminochlorotriazine ; ns , not significant individual plates were washed once with pbs , 
 and 0.5 ml of accutase was applied to detach cells from the plate s 
 surface . following a short incubation at 37 c in 5% co2 ,
the plates were retrieved , and the cells were resuspended in 10 
 ml of pbs , triturated to a single cell suspension , and transferred 
 to a 15 ml conical tube .
an aliquot of the cell suspension was removed 
 to determine cell viability using the sceptor 2.0 .
cells were exposed 
 to compounds for 6 h , and cultures were used only when no significant 
 differences in viability existed between controls and treated samples 
 and only when their viability was 85% or better .
the cell suspension 
 was subjected to centrifugation at 400 g for 5 min . 

the supernatant was aspirated from the tube , and the cell pellet was 
 lysed with the addition of a protein extraction buffer containing 
 50 mm citrate , 150 mm nacl , 0.2% np-40 , 3 mm edta , ph 4.6 , and protease 
 and phosphatase inhibitor cocktail tablets ( roche diagnostics , manneheim , 
 germany ) at recommended concentrations .
extracts were subjected to 
 centrifugation at 21  000 g for 30 min to pellet 
 cell membrane debris , and the supernatant containing cytosolic and 
 nuclear proteins was retrieved and transferred to a clean tube .
extracts 
 were snap - frozen on dry ice and placed at 80 c for storage . 

protein concentrations were determined using the modified lowry protein 
 or bsa assay kits ( thermo scientific , waltham , ma ) .
five micrograms of protein extracts from treated and untreated cell 
 lysates was subjected to sds - page gel electrophoresis under nondenaturing 
 conditions , and the separated proteins were transferred to pvdf membranes . 

membranes were blocked in phosphate buffered saline containing 0.05% 
 tween-20 ( pbst ) and 5% bovine serum albumin ( bsa ) and subsequently 
 incubated with a primary rabbit polyclonal antibody against e1 ( pw8385 , 
 enzo life sciences , plymouth meeting , pa ) .
membranes were washed three 
 times with pbst and incubated with an anti - rabbit polyclonal secondary 
 antibody conjugated to horseradish peroxidase ( hrp ) diluted in pbst 
 with 1% bsa for 1 to 2 h ( sigma - aldrich , st .
final washes 
 were performed , and ube1/e1 was visualized as two bands of approximate 
 molecular weights of 110 and 117 kda using pierce enhanced chemiluminescence 
 ( ecl ) western blotting substrate for chemiluminescence detection ( thermoscientific , 
 waltham , ma ) as directed .
hyblot cl autoradiography films ( danville 
 scientific , matuchen , nj ) were exposed to the treated blots , and developed 
 films were scanned with a gs-700 densitometer .
the data was analyzed 
 using the quantity one 1d analysis program , version 4.1 ( bio - rad , 
 hercules , ca ) .
the ratio of the activated higher molecular weight 
 ub - e1 species to the nonactivated lower molecular weight species was 
 calculated using densitometry .
comparisons of the mean ratios for 
 the two groups were performed using the unpaired t - test .
the appropriate amount of protein extract necessary 
 for detection of each protein was determined empirically using western 
 blot analysis .
briefly , protein extract ( 530 g ) was 
 diluted in lds sample buffer ( life technologies , carlsbad , ca ) containing 
 50 mm dithiothreitol ( dtt ) , heated at 70 c for 10 min , and subjected 
 to sds - page gel electrophoresis .
the separated proteins were transferred 
 to pvdf membranes ( millipore , temecula , ca ) using the semidry blotting 
 method ( invitrogen , carlsbad , ca ) , and the membranes were blocked 
 in pbs containing 0.05% tween-20 ( pbst ) and 5% bsa . the blots were 
 subsequently incubated with primary antibodies for the protein of 
 interest , including rabbit anti - e1 and mouse anti - ho-1 ( enzo life 
 sciences , plymouth meeting , pa ) .
membranes were washed three times 
 with pbst and incubated with the appropriate secondary antibodies 
 ( goat anti - rabbit - hrp or goat anti - mouse - hrp , 1/1000 dilution , sigma - aldrich , 
 st .
louis , mo ) diluted in pbst with 1% bsa for 1 to 2 h. final washes 
 were performed , and proteins were visualized and analyzed as described 
 above .
affinity binding and cross - linking of e1 protein 
 to dynabeads with protein g ( dynabeads ) ( life technologies , carlsbad , 
 ca ) was accomplished as follows : dynabeads were resuspended , and 7.5 
 mg of the bead slurry was transferred to a microfuge tube .
the beads 
 were washed three times with 500 l of 0.1 m citrate / phosphate 
 buffer , and a solution of 50 g of a rabbit polyclonal antibody 
 ( bethyl laboratories , montgomery , tx ) in 200 l of citrate / phosphate 
 buffer was prepared and added to the beads following the washes .
the 
 mixture was incubated for 3 h at 4 c to allow for affinity binding 
 of the antibody to the beads . following the incubation ,
the bead / antibody 
 mixture was washed three times with citrate / phosphate buffer and with 
 two subsequent washes with 20 mm triethanolamine , ph 8.2 ( tea ) .
subsequently , 
 250 l of a 20 mm solution of the imidoester cross - linker dimethyl 
 pimelimidate2hcl ( dmp ) ( thermoscientific ) in 20 mm tea was added , 
 and the mixture was incubated for 30 min at room temperature with 
 rocking .
the dmp solution was removed , and the cross - linked bead / antibody 
 mixture was washed for 15 min in 50 mm tris , ph 7.5 , at room temperature 
 followed by three washes with pbst and then incubation in 250 l 
 of 0.1 m glycine for 5 min at room temperature .
three more washes 
 with pbst were applied , and the bead / antibody mixture was collected , 
 resupended in 250 l of pbst , and stored until use at 4 c . 
 for the immunoprecipitation procedure ,
the prepared antibody / bead 
 mixture was resuspended , and 25 l of the slurry was transferred 
 into microfuge tubes for each sample and washed three times with 500 
 l of pbst .
the last wash was removed from the beads , and 100 
 g of cell protein extract in 250 l of pbst was added 
 to the beads .
the beads were washed three times with pbst and resuspended 
 in 20 l of 1% formic acid , the solution was evaporated using 
 the speedvac ( gmi , ramsey , mn ) , and the dried proteins were stored 
 at 4 c until processed .
to determine covalent protein modifications , 
 the dried proteins were first resuspended in a solution of 8 m urea 
 and 100 mm tris , ph 8.5 , and reduced with tcep ( 10 mm ) .
sample was 
 diluted back to 2 m urea , trypsin was added , and digestion was allowed 
 to proceed overnight at 37 c .
data on resulting peptides were 
 acquired by first separating them on a 20 cm  0.1 mm rp analytical 
 column packed into a nanospray emitter tip directly coupled to an 
 q - exactive mass spectrometry ( thermofisher ) using an aqueous ( a : 0.1% 
 formic acid ) to organic ( b : acetonitrile , 0.1% formic acid ) gradient 
 delivered by an eksigent hplc pump at 400 nl / min .
starting gradient 
 conditions were 98% a and 2% b , and after a 10 min load phase , b was 
 ramped to 40% over 70 min and was then further ramped to 95% over 
 5 min , held for 1 min , and then returned to 98% a for the remainder 
 of the 90 min cycle .
peptides were ionized directly into the mass 
 spectrometry , where both intact masses ( ms ) and fragmentation patterns 
 ( ms / ms ) of the peptides were collected .
peptide ms / ms spectral data 
 were searched against the protein database using sequest , and identifications were collated and filtered 
 using scaffold ( proteosome software inc . , portland , or ) .
protein ( 2.55.0 g ) in 5 l of lysis 
 buffer was added to 5 l of 12% sodium dodecyl sulfate and 10 
 l of 20 mm 2,4-dinitrophenylhydrazine ( dnph ) in 1,1,1-trichloroacetic 
 acid .
the solution was vortexed and incubated at 25 c for 20 
 min , and then 7.5 l of neutralizing solution was added ( 2 m 
 tris , 30% glycerol , 19% 2-mercaptoethanol ) .
negative protein controls 
 were prepared similarly except that 1,1,1-trichloroacetic acid without 
 dnph was used .
equal amounts of dnph derivatized and negative control 
 protein samples were loaded , separated by sds - page , and transferred 
 to immobilon - p membrane using an xcell ii blot module ( invitrogen , 
 carlsbad , ca ) . after blocking nonspecific binding sites ,
the membranes 
 were probed with a 1:10000 dilution of a rabbit monoclonal anti - glyceraldehyde 
 3-phosphate dehydrogenase ( gapdh ) ( 2118s , cell signaling technology 
 inc . , danvers , ma ) , and gapdh was measured by densitometry after incubation 
 using horseradish peroxidase conjugated secondary antibody ( a-8275 , 
 anti - rabbit - hrp , sigma , st .
the membranes were then probed using polyclonal rabbit anti - dnph antibody 
 ( a-6430 : molecular probes , eugene , or , dilution 1:10000 ) , and the 
 carbonylated proteins were measured using densitometry after incubation 
 with hrp - conjugated anti - rabbit secondary antibody and chemiluminescence . 

the optical density of carbonylated proteins for each sample was normalized 
 to the optical density of gapdh within the same sample .
an emulsion of ethyl arachidonate in 0.1 m phosphate 
 buffer containing 0.2% sds ( ph 7.4 ) was prepared by removing the solvent 
 from a 100 mm solution in chloroform ( 80 l ) and sonicating 
 the residue in the same buffer ( 1 ml ) for 5 min .
an aliquot ( 150 l ) 
 of the suspension was mixed with 1 mm cucl2 20 mm ascorbic 
 acid and phosphate buffer ( ph 7.4 ) with ziram or dmdc alone or combined 
 with triethylenetetramine hydrochloride ( tet ) in the same buffer . 

the final concentrations were 4 mm ethyl arachidonate , 4 mm ascorbic 
 acid , 100 m cu , 50 m ziram , 100 m 
 dmdc , and 600 m tet .
the samples were incubated at 37 c for 2 h and centrifuged 
 at 10  000 g for 10 min .
next , 100 l 
 aliquots of supernatant were mixed with 100 l of 2,6-di - tert - butyl-4-methylphenol ( bht ) in ethanol ( 20 mm ) and 800 
 l of diethyl thiobarbiturate acid ( detba ) reagent ( 100 mg of 
 detba was dissolved in 10 ml of warm ethanol and mixed with 40 ml 
 of 0.125 m phosphate buffer ( ph 3.0 ) ) and incubated at 95 c 
 for 1 h. the solutions were cooled to room temperature , then purified 
 by passing through 1 ml supelclean lc-18 spe cartridges ( supelco , 
 inc .
the residue 
 was reconstituted with 100 l of a 1:1 mix of eluent a and b. 
 eluent a was 10% acetonitrile with 0.1% triethanolamine and eluent 
 b was 100% acetonitrile .
corning , ny ) , the samples were analyzed using 
 a lichrocart 125  4 mm lichrosphere 100 rp-18 ( 5 m ) column 
 ( em science , gibbstown , nj ) running at 1 ml / min .
the elution profile 
 was 100% a going to 90% b in 7 min , holding for 5 min , and returning 
 to a in 1 min . a shimadzu lc-10ad pump connected to a sil-10ad autosampler 
 and
spd-10a uv / vis detector controlled by a slc-10a controller and 
 ezstart 7.4 software were used for the analysis . under these conditions , 

standard 
 solutions of 1,1,3,3-tetramethyoxypropane ( tmp ) , precursor to mda , 
 in a 1:1 mixture of ethanol and water were made and reacted with detba 
 reagent to generate a mda standard curve .
one hundred 
 and twenty micrograms of protein extract was digested overnight in 
 1 ml of 50% omnitrace ultra nitric acid in metal - free 
 conical tubes ( vwr , radnor , pa ) and then diluted with 9 ml of millipore 
 high - resistance purified water .
element quantification analysis was 
 performed with thermo element 2 hr - icpms ( thermo fisher scientific , 
 bremen , germany ) equipped with the esi autosampler ( elemental scientific , 
 omaha , ne ) .
the diluted acid digested samples were taken up through 
 automated aspiration via a 0.50 mm i.d .
elements of interest were measured on isotopes mn , fe , cu , and zn at medium resolution 
 ( r = 4300 ) to separate any potential molecular interference .
one - tailed and two - tailed student s t tests , one - way anova , tukey s multiple comparison 
 test , and dunnett s test were performed using prism 4 ( graphpad 
 software , inc . ) . for
proteomics expression comparisons , decyder software , 
 version 6.5 ( ge healthcare bio - sciences ) was used .
previous 
 studies demonstrated that e1 was covalently modified at 
 reactive cysteines within the ubiquitin - binding groove in brain tissues 
 of rats treated with dedc and in hek293 cell extracts exposed to thiol - reactive 
 electrophiles . to determine
if e1 is a target 
 for electrophilic adduction by other dithiocarbamates , we used an 
 in vitro cell culture exposure assay followed by western blot analysis 
 to quantify ub - e1 .
we used hek293 cells because they offer a robust 
 human cell line amenable to studying the multiple incubation conditions 
 employed in this study and because they are a previously established 
 model for evaluating covalent adduction of e1 by thiol - reactive electrophiles 
 and ziram - mediated inhibition of e1 .
subsequent 
 to establishing a decrease in ub - e1 in our samples , e1enriched 
 cell extracts were subjected to lc / ms / ms for detection of dithiocarbamate - derived 
 adducts .
we utilized two dithiocarbamates , ziram and dmdc ( scheme 1 ) , in these studies , as the structure and potential 
 adducts formed by these compounds would be comparable to those for 
 dedc .
ziram at 10 m produced a 
 75% reduction in ub - e1 , and 20 m dmdc significantly decreased 
 ub - e1 to almost half that of the controls ( figure 1 ) ; ziram also significantly inhibited e1 at 1 m ( supporting information ) . to assess whether altered 
 expression of e1 contributed to the changes in the ratio of ub - e1 
 to e1 , we determined total e1 levels in our treated cells and found 
 that they remained unchanged ( data not shown ) . to determine if covalent 
 modification occurred on the reactive e1 cysteines identified for 
 dedc in vivo , we searched for covalent modifications in e1-enriched 
 hek293 extracts using lc / ms / ms . on the basis of the structures of 
 ziram and dmdc and the previous adduct identified for dedc ,
we analyzed 
 for an s - methylaminocarbonyl adduct corresponding 
 to a mass of + 58 da on all peptides identified in the digests ( typically , 
 7080% sequence coverage was obtained ) .
however , in contrast 
 to our in vivo studies , this adduct was not detected in the cell extracts 
 of the treated hek293 cells ( data not shown ) .
to evaluate the potential 
 of glutathiotinylation , we also searched for + 305 da modifications , 
 but no evidence for this modification was observed .
this suggests 
 that significant e1 inhibition was produced by ziram and dmdc either 
 at levels of e1 adduction that were not detectable by lc / ms / ms or 
 through a mechanism independent of cysteine adduction .
the shorter 
 exposure duration used in the cell cultures or a difference in the 
 capacity of the hek293 cells to bioactivate these dithiocarbamates 
 may have contributed to this observation . additionally ,
although dmdc 
 and dedc are structurally very similar and subject to the same metabolic 
 pathways , a smaller contribution from the bioactivation pathway leading 
 to adduct formation in dmdc s biotransformation can not be ruled 
 out from this study . while these experimental limitations may be responsible 
 for the lack of detectable adducts , the results highlight the possibility 
 that other cellular mechanisms contribute to our observed inhibition 
 of e1 .
earlier studies in cell models suggest that e1 activation is 
 redox sensitive , and in vivo studies have demonstrated that dithiocarbamates 
 complex metals and cross cell membranes , potentially increasing intracellular 
 levels of redox active metals .
thus , we reasoned that exposure 
 to ziram and dmdc may be contributing to the observed e1 inhibition 
 by promoting intracellular oxidation , a mechanism that could potentially 
 be shared by many environmental agents and disease conditions .
inhibition of e1 activation by 10 m ziram ( a ) and 20 m 
 dmdc ( b ) in the presence and absence of 60 m tet .
representative 
 western blots show activated ( ub - e1 ) and nonactivated ( e1 ) species 
 of e1 ubiquitin activating enzyme separated by sds - page .
graphs depict 
 the mean ratio of ub - e1/e1 values ( + se ) for each treatment group . 

statistical comparisons were performed using one - way anova followed 
 by tukey s multiple comparison test .
previous studies reported e1 activity to be reduced in states 
 of 
 oxidative stress , and certain dithiocarbamates have been shown to 
 complex and transport redox active copper into cells . to explore the possibility that decreased ub - e1 levels in our cell 
 culture assay resulted from an increase in intracellular copper , we 
 exposed cells to 10 m ziram or 20 m dmdc in the presence 
 of a known copper chelator , tet , and determined ub - e1 levels ( figure 1 ) . sixty micromolar
tet significantly mitigated 
 the decrease of ub - e1 due to ziram exposure but did not completely 
 prevent decreased ub - e1 levels when coincubated with dmdc .
subsequent 
 studies using coincubation with 120 m tet did maintain ub - e1 
 levels similar to that of the control in dmdc - treated samples ( data 
 not shown ) .
thus , we chose to continue our experiments utilizing 60 
 m tet , as this was sufficient to maintain ub - e1 levels with 
 ziram treatment and minimize loss of ub - e1 in dmdc - treated samples . 

we next determined the changes in intracellular levels of copper and 
 zinc , produced by ziram or dmdc , with and without tet .
our results 
 show that copper levels were significantly elevated in cells treated 
 with either dithiocarbamate alone , albeit to different extents ( figure 2 ) . in ziram - treated samples ,
copper levels were 
 3-fold higher than that in control , whereas dmdc produced a smaller 
 but significant increase .
zinc measurements revealed an approximately 
 2-fold increase in intracellular zinc for ziram , with no significant 
 change for dmdc treatment .
thus , although the incubation conditions 
 exposed the cells to molar equivalents of n , n - dimethyldithiocarbamate , the zinc complexed formulation 
 was more effective in transporting copper and zinc intracellularly 
 and inhibiting e1 than the sodium salt .
intracellular zinc and 
 copper levels in samples treated with 10 
 m ziram ( a ) and 20 m dmdc ( b ) in the presence and absence 
 of 60 m tet .
statistical comparisons 
 were performed using one - way anova followed by dunnett s multiple 
 comparison test .
although 60 m 
 tet prevented intracellular copper transport 
 by dmdc , it had no effect on intracellular copper levels in ziram - treated 
 cells .
therefore , it appeared that the ability of tet to decrease 
 ziram - mediated inhibition of e1 was not entirely dependent on preventing 
 intracellular transport of copper or zinc . to address the possibility 
 that tet maintains e1 activity through moderating dithiocarbamate - promoted 
 oxidative injury , we determined the ability of tet to decrease ziram- 
 and dmdc - mediated lipid peroxidation , protein oxidative damage , and 
 expression of heme oxygenase-1 ( ho-1 ) .
for lipid peroxidation , we 
 utilized a previously established in vitro system composed of an emulsion 
 of ethyl arachidonate incubated with cucl2 in the presence 
 of either ziram or dmdc , with or without tet , and measured production 
 of mda . both ziram and dmdc
significantly 
 increased mda , and similar to intracellular transport of copper , ziram 
 was more effective in generating mda ( figure 3 ) .
coincubation with tet prevented mda production for both compounds . 
 to evaluate protein oxidative damage , we measured protein carbonyls 
 in hek293 cells .
both ziram and dmdc produced a significant increase 
 in the levels of protein carbonyls relative to controls , and tet was 
 effective in preventing the elevated protein carbonylation produced 
 by both dithiocarbamates ( figure 4 ) .
thus , 
 although tet did not completely prevent the intracellular transport 
 of copper , it did diminish the oxidative stress associated with dithiocarbamate 
 exposure , possibly through sequestering copper and reducing the level 
 of dithiocarbamate copper complexes .
mda production by ziram and dmdc in the 
 presence and absence of 
 tet .
mda levels were determined in an emulsion of 4 mm ethyl arachidonate , 
 4 mm ascorbic acid , 100 m cucl2 treated with 50 
 m ziram , or 100 m dmdc with and without 600 m 
 tet as well as in controls incubated similarly without dithiocarbamate 
 or tet .
statistical comparisons were performed using one - way anova 
 followed by dunnett s multiple comparison test .
carbonylated protein determinations in samples treated 
 with 10 
 m ziram and 20 m dmdc in the presence and absence of 
 60 m tet .
protein samples were obtained from control cells 
 incubated with dmso vehicle and exposed cells incubated with either 
 10 m ziram or 20 m dmdc in the presence and absence 
 of 60 m tet , reacted with 2,4-dinitrophenylhydrazine , and quantified 
 by western blot using an anti 2,4-dnp antibody . mean optical density 
 ( od ) values ( + se ) , expressed as a ratio to gapdh as an intra sample 
 standard and then normalized to control mean values , are shown .
statistical 
 comparisons were performed using one - way anova and dunnett s 
 multiple comparison test or two - way unpaired student s t test ( control vs tet ) .
protein expression levels are regulated by the antioxidant 
 response element , have been shown to increase from dithiocarbamate 
 exposure , and can be used as a marker for the cellular response to 
 oxidative stress .
however , while the addition of 60 m tet did not significantly 
 reduce this effect for ziram , coincubation with 120 m tet did 
 significantly decrease ho-1 levels relative to ziram alone . because 
 transcriptional regulation of ho-1 includes participation of a metal 
 response element present in the 5 regulatory region of the 
 gene in addition to the antioxidant response element , the observed 
 ho-1 expression for ziram likely reflects the elevated levels of copper 
 and zinc that remained in the tet and ziram - treated cells .
protein expression 
 levels of heme oxygenase-1 ( ho-1 ) in cells treated 
 with 10 m ziram ( a ) and 20 m dmdc ( b ) in the presence 
 and absence of tet .
results are shown as the mean ( + se ) ratio to gapdh within 
 the same samples .
statistical comparisons were performed by one - way 
 anova and tukey s multiple comparison test ; n = 5 for all groups . to delineate the relative contributions of copper and zinc 
 to e1 
 inhibition ,
we performed experiments under metal - free media incubation 
 conditions in which copper and zinc levels could be modulated independently . 

cells maintained in emem were washed with pbs , incubated in hank s 
 balanced salt solution ( hbss ) , and exposed to ziram or dmdc in the 
 presence and absence of added zncl2 or cucl2 and to either zncl2 or cucl2 alone . under 
 these conditions , all test compounds significantly decreased ub - e1 
 levels relative to that in controls , and combining dmdc with either 
 zncl2 or cucl2 resulted in a further decrease 
 of ub - e1 relative to that in cells treated with dmdc alone ( figure 6 ) . in the lysates obtained from cells incubated 
 with ziram combined with cucl2 , neither a band for ub - e1 
 nor e1 could be detected by western blot . under these metal - free incubation 
 conditions ,
ziram still significantly increased intracellular zinc 
 and copper , whereas dmdc did not significantly change either metal 
 ( figure 7 ) .
the zinc contained in ziram can 
 account for the elevation of zinc in this incubation system , but the 
 ability of ziram to elevate copper demonstrates the greater efficiency 
 of ziram relative to dmdc to either scavenge trace quantities of copper 
 or to mobilize copper located within the cell membrane .
the ability 
 of zncl2 or cucl2 alone to inhibit e1 and to 
 enhance the effect of both dithiocarbamates is consistent with the 
 elevation of either zinc or copper contributing to inhibition of e1 . 

thus , the relative efficacy of ziram and dmdc to elevate intracellular 
 levels of these metals could account for their relative potency for 
 e1 inhibition .
however , considering the data obtained from coincubation 
 of the dithiocarbamates with tet , the elevation of zinc or copper 
 alone is not sufficient but also requires that the metals be in a 
 redox - active form accompanied by increased oxidative stress .
an unexpected 
 finding was the lack of a significant change in intracellular copper 
 for dmdc even though it produced a significant decrease in ub - e1 . 

although it can not be determined from the current study , dmdc may 
 have produced a small but biologically relevant increase in copper 
 that was not detectable in our methodology or it may have shifted 
 a fraction of the protein - associated intracellular copper to a more 
 redox - active form , e.g. , a dmdc copper complex .
cells were exposed to 10 m ziram , 20 m cucl2 , 20 m zncl2 , 20 m dmdc , 20 m dmdc 
 plus 20 m cucl2 , or 20 m dmdc plus 20 m 
 zncl2 .
graphs depict the mean ratio of ub - e1/e1 values 
 ( + se ) for each treatment group as the percent of control .
statistical 
 comparisons were performed using one - way anova followed by tukey s 
 multiple comparison test . in samples incubated with 10 m ziram 
 plus 20 m cucl2 , bands were not detectable for either 
 ub - e1 or e1 .
intracellular copper 
 and zinc levels in hbss under metal - free culture 
 conditions .
cells were exposed to 10 m ziram or 20 m 
 dmdc , 20 m cucl2 , 20 m zncl2 , 
 10 m ziram plus 20 m cucl2 , 20 m dmdc 
 plus 20 m cucl2 , or 20 m dmdc plus 20 m 
 zncl2 .
statistical comparisons 
 were performed using one - way anova followed by tukey s multiple 
 comparison test .
the studies presented 
 here support intracellular transport of copper 
 and zinc accompanied by oxidative stress as a mechanism for dithiocarbamate - mediated 
 e1 inhibition .
these data suggest that e1 is a common target of other 
 environmental toxins that can perturb the redox status of the cell . 
 to further evaluate the potential of our cell culture assay and the 
 use of monitoring e1 function as a generalized response of exposure 
 to environmental toxins , we evaluated ub - e1/e1 ratios in samples treated 
 with a series of compounds , including additional dithiocarbamates , 
 thiocarbamates , triazines , benzimadazoles , and several metals ( table 1 ) . among the compounds tested , seven of the pesticides 
 produced a significant decrease in ub - e1 , and four produced a significant 
 increase in activated e1 when compared to that in the control .
we 
 also observed a significant increase in ub - e1 with exposure to fe(clo4)2 and 4-hydroxynonenal as well as a significant 
 decrease in ub - e1 by mncl2 and the e1 specific inhibitor , 
 pyr-41 , compared to that in control samples .
it is interesting to 
 note that while the majority of the compounds that altered e1 function 
 did so by inhibiting its activity , some compounds and one metal increased 
 ub - e1 . while , to date , our investigations have supported two putative 
 mechanisms by which e1 activity may be altered , the modulation of 
 e1 activity by the compounds in table 1 suggests 
 that additional mechanisms may be elucidated through continued analysis 
 of altered e1 function .
collectively , our results are consistent 
 with e1 regulating downstream 
 targets , at least partially , in response to intracellular levels of 
 ros .
e1 inhibition increased 
 when the antioxidant capacity was diminished in pc12 cells through 
 depletion of glutathione , and upon reestablishment of normal levels 
 of glutathione , e1 activity returned .
more recent 
 studies in yeast cultures also showed that reactive cysteines on e1 
 and the e2 conjugase cdc34 were redox - sensitive and that , when oxidized , 
 they effectively sequestered the proteins catalytic cysteines , 
 leading to downregulation of cell cycle events dependent on e1 ubiquitination 
 and cdc34 function . thus , it is possible 
 that the redox sensitivity of e1 observed in our studies is an integral 
 part of its function and promotes maintenance of cellular homeostasis 
 through its responsiveness to cellular oxidation states .
numerous 
 studies have demonstrated the importance of ubiquitin 
 regulation to the development and function of the central nervous 
 system .
the importance of e1 activity in nervous system function is 
 underscored by the fact that mutations in e1 rarely result in viable 
 offspring and infants that survive do so briefly and suffer from x - linked 
 infantile spinal muscular atrophy ( xl - sma ) . identifying e1 as being susceptible to perturbation by environmental 
 toxins lends support to the premise established by multiple epidemiological 
 studies that environmental pesticides are potential contributors to 
 the etiology of neurodegenerative disease .
in fact , the singular nature 
 and apical positioning of e1 in the cascade of ubiquitin - regulating 
 events make it a strong candidate as a gateway molecule for environmental 
 perturbation of cellular function .
furthermore , e1 susceptibility 
 in concert with increased sensitivity of gene variants of ups - related 
 proteins , as reported in rhodes et al . , could potentiate or compound 
 the individual contribution of each of these biological factors . the studies reported here support this premise 
 and suggest that the redox - sensitive nature of e1 may be an essential 
 part of its biological function as well as contribute to its vulnerability 
 to deregulation by environmental agents .
continued assessment using 
 both in vivo and in vitro models is essential to further our understanding 
 of how alterations in e1 activity produced by environmental factors 
 can influence biological systems and contribute to neurological disease .
 ubiquitin activating enzyme e1 plays 
 a pivotal role in ubiquitin 
 based protein signaling through regulating the initiating step of 
 the cascade . 
 previous studies demonstrated that e1 is inhibited by 
 covalent modification of reactive cysteines contained within the ubiquitin - binding 
 groove and by conditions that increase oxidative stress and deplete 
 cellular antioxidants . in this study , we determined the relative contribution 
 of covalent adduction and oxidative stress to e1 inhibition produced 
 by ziram and sodium n , n - dimethyldithiocarbamate 
 ( dmdc ) in hek293 cells . 
 although no dithiocarbamate - derived e1 adducts 
 were identified on e1 using shotgun lc / ms / ms for either ziram or dmdc , 
 both dithiocarbamates significantly decreased e1 activity , with ziram 
 demonstrating greater potency . 
 ziram increased intracellular levels 
 of zinc and copper , dmdc increased intracellular levels of only copper , 
 and both dithiocarbamates enhanced oxidative injury evidenced by elevated 
 levels of protein carbonyls and expression of heme oxygenase-1 . to 
 assess the contribution of intracellular copper transport to e1 inhibition , 
 coincubations were performed with the copper chelator triethylenetetramine 
 hydrochloride ( tet ) . 
 tet significantly protected e1 activity for both 
 of the dithiocarbamates and decreased the associated oxidative injury 
 in hek293 cells as well as prevented dithiocarbamate - mediated lipid 
 peroxidation assayed using an ethyl aracidonate micelle system . because 
 tet did not completely ameliorate intracellular transport of copper 
 or zinc for ziram , tet apparently maintained e1 activity through its 
 ability to diminish dithiocarbamate - mediated oxidative stress . 
 experiments 
 to determine the relative contribution of elevated intracellular zinc 
 and copper were performed using a metal free incubation system and 
 showed that increases in either metal were sufficient to inhibit e1 . 
 to evaluate the utility of the hek293 in vitro system for screening 
 environmental agents , a series of additional pesticides and metals 
 was assayed , and eight agents that produced a significant decrease 
 and five that produced a significant increase in activated e1 were 
 identified . 
 these studies suggest that e1 is a sensitive redox sensor 
 that can be modulated by exposure to environmental agents and can 
 regulate downstream cellular processes . 
Introduction
Materials and Methods
Results and Discussion