the effects of the exposure to extremely low frequency electromagnetic fields ( elf - emfs ) , generated by power lines and household electric appliances , have been receiving increasing interest for the feasible harmful effects on human health . the abovementioned human - made fields have a different wave form , coherence , and frequency with respect to natural emfs , and their effects on living biological systems are far from being completely understood . substantially , human - made emfs are above the naturally occurring electric and magnetic fields . the average b value of the static geomagnetic field is 40  10   t approximately     [ 1 , 2 ] .       a variety of biological hazards , partly attributable to an increased level of free radicals , were associated with the exposure to elf - emfs     [ 35 ] . in cellular models , a significant postexposure increase in reactive oxygen species ( ros ) has been reported , suggesting that elf - emfs are able to induce the activation of cellular processes in the investigated models     [ 4 , 5 ] . additionally , elf - emfs were likely to cause an increase in free radicals lifetime in cells     [ 6 , 7 ] . besides that , elf - emfs have been shown to alter second messenger pathways such as calcium signalling     [ 1 , 8 ] , thus leading to downstream physiological effects . in fact , 50 hz emfs have been reported to produce an increase in the intracellular calcium level in the pituitary corticotrope - derived att20 d16 v cells , and a consequent rearrangement of the cytoskeletal network     . several epidemiological studies reported evidences suggesting a link between elf - emfs exposure and cancer risk     [ 1 , 10 , 11 ] . these observations were supported by cellular studies reporting the electromagnetic field - induced effects on biological systems , most notably on the activity of components implicated in the regulation of cell proliferation     [ 1 , 12 , 13 ] .      nevertheless , it is still unclear whether and how the exposure to elf - emfs promotes carcinogenesis . mutagenic effects have been extensively described , such as a global increase in the frequency of chromosomal aberrations , in micronuclei formation , or in dna strand breaks .        only limited data are currently available on the specific effects of elf - emfs on enzyme functionality , although it is believed that they can affect enzyme activity     [ 1 , 7 , 13 , 1720 ] . in this perspective , our attention has been focused on the effects of elf - emfs exposure on enzymes that regulate cellular homeostasis , the proteasome in particular . tumor suppressors , cell - cycle regulators , transcription factors , and antiapoptotic proteins are examples of proteasome substrates     [ 2123 ] . the cyclin dependent kinase p27 , known as a pro - apoptotic protein , is an important regulator of the cell cycle progression , controlling the g1/s transition , where it reaches the highest concentration . its cellular levels are modulated by the proteasome - dependent degradation , and its decrease has been related to an activation of the proteasome functionality , preventing the cell from undergoing apoptosis .       the 20s proteasome plays a central role in the cellular antioxidant defence , since partially folded , unfolded , and oxidized proteins     [ 2528 ] , as well as proteins with the reduction of disulfide bonds , are susceptible to proteasomal degradation . the 20s proteasome is a member of the n - terminal nucleophile hydrolases family ( ntn - hydrolases ) , a class of enzymes that catalytically use an n - terminal residue as both a polarizing base and a nucleophile     . in eukaryotic proteasomes only three  subunits ( 1 , 2 , and 5 ) are catalytically active , each linked to a specific catalytic component : the  peptidylglutamyl - peptide hydrolizing ( t - l , cleaving after basic residues ) , and the  chymotrypsin - like  ( cht - l , cleaving after hydrophobic residues ) activities , respectively     [ 3032 ] . in addition , two more catalytic activities , namely , the braap ( cleaving bonds after branched chain and aromatic amino acids ) and the snaap ( cleaving after small neutral amino acids ) , were reported     .      among the modulators of the 20s proteasome activity , the polyphenolic compound epigallocatechin-3-gallate ( egcg ) , the major component of green tea , has been demonstrated to possess a strong free radical scavenging effect in several cell lines     [ 3439 ] . considering the key role of the proteasome in the removal of oxidized proteins and in the regulation of the cell cycle progression , in the present paper we have investigated the effect of elf - emfs on the 20s proteasome functionality . furthermore , egcg protective action towards elf - emfs mediated oxidative damage has been also evaluated . the oxidized protein detection kit was purchased from appligene - oncor ( strasbourg , france ) . substrates for assaying the cht - l , t - l and pgph activities ( suc - leu - leu - val - tyr - amc , z - leu - ser - thr - arg - amc , and z - leu - leu - glu - amc ) were purchased from sigma - aldrich s.r.l . ( milano , italy ) . the z - gly - pro - ala - leu - ala - pab substrate was the kind gift of professor m. orlowski ( department of pharmacology , 12-o - tetradecanoylphorbol-13-acetate ( tpa ) and ( -)epigallocatechin-3-gallate ( egcg ) were purchased from sigma - aldrich s.r.l . egcg was used directly without further purification and was dissolved in assay buffer ( tris - hcl 50 mm , ph 8.0 ) .      the mouse antihuman p27   polyclonal antibody was obtained by inalco s.p.a . the rabbit anti - gapdh ( fl-335 ) polyclonal antibody was purchased from santa cruz biotechnology inc . sigmamarker , wide molecular weight range protein standards for sds - page were obtained from sigma - aldrich s.r.l . the human colon adenocarcinoma cell line caco 2 ( istituto zooprofilattico sperimentale lombardia ed emilia romagna , italy ) was cultured in d - mem medium ( gibco invitrogen cell culture , milano , italy ) supplemented with 15% fetal calf serum , 0.4% l - glutamine , 1% non essential amino acids , penicillin , and streptomycin . cells were grown in 100 mm tissue culture dishes at an initial concentration of 4  10 cells / ml in a total volume of 15 ml . after a 24-hour starvation without serum , cells were continuously exposed to a sinusoidal 50 hz magnetic field at a flux density of 1 mt ( rms ) in a solenoid for 24 , 48 , and 72 hours . control samples were placed under the same conditions in a solenoid with no em field applied . the stray field inside the nonpowered solenoid was   < 0.5 t . both samples were placed into a water - jacketed , thermo - regulated and atmosphere - controlled incubator ( 37.0  0.5c and 5% co2 ) . caco cells were cultured in the presence and absence of 150 m egcg and in the presence and absence of 0.1 m tpa     .       after removing the medium and washing with cold pbs after centrifuging at 1600 rpm for 5 minutes , the pellet was homogenized in a lysis buffer ( 20 mm tris , ph 7.4 , 250 mm sucrose , 1 mm edta , and 5 mm -mercaptoethanol ) and passed through a 25-gauge needle for at least ten times . lysates were centrifuged at 12000  g   for 15 minutes , and the supernatants were stocked at   80c . protein concentration in cell lysates was determined by bradford method   using bovine serum albumine as a standard . elf - emfs were generated by a 20 cm diameter      40 cm length solenoid . the solenoid was wound in three forward - backward - forward continuous layers with 600 turns of 2 mm diameter copper wire . it was driven by the 50 hz power line through with a variable autotransformer and generated a magnetic flux density of 1 mt ( rms ) for an applied voltage of 6 v ( rms )     [ 9 , 41 ] . the solenoid was positioned into a water - jacketed temperature and atmosphere regulated incubator ( 37.0  0.5c and 5% co2 ) . the dishes were placed horizontally into the solenoid , so that the magnetic flux was parallel to the bottom . moreover , dishes were placed in a volume determined by measurements of field variations inside the solenoid . in fact , field intensity , measured with a calibrated chauvin arnoux gaussmeter , was within   5% of centre value inside the cylindrical exposure volume of 11 cm by 17 cm along the solenoid axis . cells were exposed in continuous to a 50 hz magnetic field at a flux density of 1 mt ( rms ) for 24 , 48 and 72 hours . control samples were placed in a solenoid with no field applied positioned into a second identical water - jacketed temperature and atmosphere regulated incubator . the stray field inside the nonpowered solenoid was < 0.5 t .      direct monitoring of sample temperatures before , during , and after exposure for both the control and exposed conditions was performed in continuous using a digital multimeter ( metex mod . m-3630d ) controlled by scopeview software ( vers . 1.06 ) for data logging ( the acquisition was carried out at 0.0028 hz ) . the temperature probe was placed within the centre of the solenoid ( either energized or not ) in a culture dish containing medium with no cells : no significant temperature difference between culture media of exposed or unexposed dishes was revealed . cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) conversion as previously described     [ 42 , 43 ] . briefly , following experimental treatment , cells were washed in phosphate - buffered saline solution ( pbs , ph 7.5 ) , and then mtt ( final concentration 0.5 mg / ml ) was added to culture medium without fetal calf serum and incubated for 2 hours at 37c . the cht - l , t - l , pgph , and braap activities in cell lysates were determined with a shimadzu rf5300 spectrofluorometer as previously reported     [ 33 , 44 , 45 ] , using 5 m suc - leu - leu - val - tyr - amc , 5 m z - leu - ser - thr - arg - amc , 5 m z - leu - leu - glu - amc , and 0.5 mm z - gly - pro - ala - leu - ala - pab , respectively , as substrates . cell lysates ( 5 g total proteins ) were incubated with the synthetic substrates in the assay buffer ( 50 mm tris - hcl , ph 8.0 ) up to a final volume of 500 l . after 1 hour of incubation at 37c , the measurement of the hydrolyzed 7-amino-4-methyl - coumarin ( amc ) or 4-aminobenzoic acid ( paba ) was detected ( amc : exc = 365 nm , em = 449 nm ; paba : exc = 304 nm , em = 664 nm ) .        since the braap component cleaves exclusively the leu - ala bond in z - gly - pro - ala - leu - ala - pab , a coupled two - step reaction in the presence of excess aminopeptidase n ( apn ) , 20 g of protein , ( ec 3.4.11.2 ) ,     , was used for the determination of the activity . aminopeptidase n was purified from pig kidney , as reported elsewhere     [ 46 , 47 ] .       with the aim of evaluating the 20s proteasome contribution to the short peptide cleavage , control experiments were performed in the presence of specific inhibitors : z - gly - pro - phe - leu - cho ( 5 m ) for the braap activity and lactacystin ( 5 m ) for the cht - l component     [ 48 , 49 ] . immunoblot detection of carbonyl groups was performed with the oxyblot oxidized protein detection kit ( appligene - oncor , strasbourg , france ) , according to the manufacturer . briefly , 15 g of cell lysates were incubated for 15 minutes at room temperature with 2,4-dinitrophenylhydrazine to form the dinitrophenylhydrazone carbonyl derivate and separated on a 12% sds / page . the modified proteins were then   transferred to a polyvinylidene fluoride membrane ( millipore corporation , bedford , ma , usa ) and revealed by antidinitrophenylhydrazone antibodies as specified in the oxyblot data sheet . core  subunits of the human 20s proteasome ( 1 : 1000 ) were carried out in order to reveal differences in the expression levels of the complex in cell lysates . immunoblot experiments using anti - p27 human and mouse ( mouse ) monoclonal antibody ( 1 : 100 ) were performed by electroblotting 30 g lysates samples per lane . each gel was loaded with molecular weight markers including proteins with mw from 6.5 to 205 kda ( sigmamarker wide molecular weight range , sigma - aldrich s.r.l . ) . after immunostaining , the two protein bands ( ~50 and 70 kda ) were identified overlapping the film with the comassie - brilliant blue - stained membrane .   as a control for equal protein loading glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was utilized : membranes were stripped and reprobed for gapdh using a polyclonal antibody diluted 1 : 500 ( anti - gapdh ( fl-335 ) rabbit polyclonal antibody from santa cruz biotechnology inc . ) . the immunoblot detections were carried out with enhanced chemiluminescence western blotting analysis system ( ge - healthcare , milano , italy ) using peroxidase conjugated antimouse and antirabbit secondary antibodies . each western blot film was scanned ( 16 bits greyscale ) , and the obtained digital data were processed to calculate the background mean value and its standard deviation . the background - free image was then obtained subtracting the background intensity mean value from the original digital data . the integrated densitometric value associated to each band was then calculated as the sum of the density values over all the pixels belonging to the considered band having a density value higher than the background standard deviation . the band densitometric value was then normalized to the relative gapdh signal intensity . the ratios of band intensities were calculated within the same western blot . all the calculations were carried out using the matlab environment ( the mathworks inc . , ma , usa ) . all data are expressed as mean values      se of results obtained from six independent experiments , in which all the activities measurements were carried out in triplicate , whereas for densitometric analysis six blots were considered . analysis of variance ( anova ) was used to assess differences among multiple sets of data obtained with untreated and treated cells at different times of exposure . when significant values were found   ( p < .05 ) , post hoc comparisons of means were made using the bonferroni - dunn test . it is widely accepted that elf - emfs can influence several biological functions of cells and tissues , modulating intracellular ros levels and the cell cycle progression     [ 1 , 35 , 12 , 13 ] . the aim of this study was to investigate the effect of elf - emfs on cultured cells focalizing the attention on the 20s proteasome functionality , the main responsible for the degradation of oxidized proteins . exposing caco cells to 1 mt , 50 hz elf - emfs , resulted in a time - dependent increase in cell proliferation rate compared to unexposed control cells . in detail , at 24 hours a 1012% increase in cell proliferation rate upon elf - emfs exposure was evident and after 48 hours there was an increase from 12 to 16% . cell viability was essentially not affected by elf - emfs exposure , however several changes in the cytoplasmic proteolytic pathway have been highlighted . results on cell proliferation are shown in figure 2 , where each data point is reported as percent of control unexposed cells within the same time exposure . stimulating the cells with 0.1 m tpa , a tumor promoter able to activate protein kinase c in vivo and in vitro , caused a slighter increase in cell proliferation ( from 8 to 10% at 24 hours and from 11 to 13% at 48 hours ) and essentially no change in cell viability , as measured by the mtt reduction assay . viability in exposed cells was not less than 85% of the respective controls ( data not shown ) .       in order to test whether elf - emfs exposure causes an increase in cellular oxidative stress , thus favouring changes in protein structures , immunoblot analyses of protein carbonyl groups as shown in figure 3 , the exposure to elf - emfs led to a time dependent increase in protein oxidation . particularly evident was the increase in carbonyl groups after elf - emfs exposure and in the presence of tpa , especially at 72 hours treatment ( 1.67-fold increase , p < .05 ) ; see figure 3 . numerous publications reported on the ability of egcg to act as a free radical scavenger     [ 34 , 3639 , 52 ] . the protective action of egcg against the elf - emfs induced oxidative stress was assayed adding this polyphenol directly to the culture medium during exposure . the treatment with 150 m egcg caused , essentially , no change in cell viability as measured by the mtt reduction assay ( data not shown ) . as shown in figure 4 , the enhancement in protein carbonyl groups , evident in cells exposed to the fields , was lowered in egcg treated cells , demonstrating that egcg protected cells from protein oxidation induced by elf - emfs exposure .       as abovementioned , the 20s proteasome is the proteolytic system involved in the removal of oxidatively modified proteins     [ 2528 ] . the enzyme functionality was measured monitoring the cleavage of short synthetic peptides in whole cell extracts ; in details , the cht - l , t - l , braap , and pgph activities were assayed . the results obtained from the activity assays demonstrated that the most susceptible components to the fields were the cht - l , t - l , and pgph . in particular , the t - l and pgph components presented a very similar behaviour . in fact , in the absence of tpa , the exposure to electromagnetic fields led to the activation of both the components with a 1.16-fold increase after 24 hours treatment ( p < .05 ) . the cht - l activity presented the maximum activation at 72 hours ( 1.17-fold increase , p < .05 ) ; see figure 5 . an evident activation of the components was also manifested after the simultaneous exposure to tpa . in detail , at 72 hours a 1.18-fold , a 1.2-fold and a 1.17-fold increase was evident for the t - l , pgph , and cht - l activity , respectively . interestingly , the braap component presented a peculiar behaviour since it was not affected at 24 hours and 48 hours , while a 1.25-fold increase was evident at 72 hours exposure ( p < .05 ) ; see figure 5(b ) .        in a recent study we have shown that egcg has , besides its scavenging and antioxidant properties , a direct inhibitory effect on the 20s proteasome     . when cells were exposed to the fields in the presence of egcg the abovedescribed proteasome activation was blocked as shown in figure 6 for the cht - l activity . it is reasonable that the egcg - dependent decrease in oxidized species results in a minor exposure of protein hydrophobic residues generating an environment not favorable for the proteasome to be activated .       in order to evaluate whether the differences observed in proteasome functionality were due to changes in 20s proteasome expression levels , we performed immunoblottings using an anti-20s proteasome antibody . as shown in figure 7 , no differences were detected in the proteasome content in lysates obtained from treated and control cells , suggesting that the fields do not affect proteasome expression . the protein p27 is a cell cycle regulator with a pro - apoptotic effect and is also known as a 20s proteasome substrate . the immunoblot analysis performed with an antibody specific for this protein , showed a time dependent decrease of p27 expression following elf - emfs exposure . in the absence of tpa , a gradual decrease of p27 levels the presence of tpa led to an overall decrease of protein levels , detectable both in control and elf - emfs exposed cells . moreover , p27 levels in elf - emfs treated cells were a 10% lower than in unexposed control cells ( figure 8) . previous findings suggest that conditions of mild oxidative stress , causing a rearrangement of cellular proteins structures with the consequent exposure of normally hidden hydrophobic residues , may induce an activation of the proteasome functionality     [ 53 , 54 ] . considering this , the enhancement observed in the 20s proteasome activity , being not associated to an upregulation of its synthesis , as demonstrated by the 20s immunoblots , could be a consequence of the increase in the intracellular oxidative stress . it is possible that in order to counteract the toxicity associated to protein oxidation , cells promote the proteasome - mediated proteolytic pathway . furthermore , this increased activity could be related to the upregulation of the intracellular ca levels . an increase in [ ca ] in cells upon elf - emfs exposure was reported by numerous groups     [ 55 , 56 ] , and it is known that this divalent cation is able to modulate proteasome activity , as suggested by reshetnyak et al . regarding the cht - l component . the proteasome activation could be associated with a change in the 20s structure with the adoption of an ordered 7-fold symmetric pore conformation stabilized by interactions among a cluster of highly conserved residues ( tyr8 , asp9 , pro17 and tyr26 )     . this change lets the proteasome achieve an open conformation thus allowing the substrate to easily enter the proteolytic cavity . in fact , sds and ca behave in a similar way toward proteasome activation      and it has been shown that sds is able to increase proteasome vmax . upon elf - emfs exposure there was a decrease in p27 expression levels most likely because of the activation of the 20s proteasome . apoptosis is a normally occurring process which is used as a protective mechanism to remove damaged cells ; therefore the observed downregulation of p27 expression , induced by both elf - emfs and tpa , may prevent the cells from utilizing that strategy in order to get rid of altered cells     [ 60 , 61 ] . elf - emfs effects are potentiated by stimulation with tpa , confirming the generally accepted notion that elf - emfs behave as a tumor promoter but not as a tumour initiator     [ 1 , 62 ] . in fact , it has been published that low tpa concentrations render the cells susceptible to other stimuli , and consequently the simultaneous exposure to elf - emfs lets the two factors act in a synergistic manner . it is interesting to note that intracellular calcium concentration fluctuates upon elf - emfs exposure , affecting protein kinases activities and activating nuclear promoters . recently , it has been demonstrated that [ ca ] is able to trigger ubiquitination of endoplasmic reticulum key receptors , such as inositol 1,4,5-tris - phosphate receptor . this receptor binds to the antiapoptotic protein bcl - x(l ) , resulting in reduced endoplasmic reticulum ca concentration , increased oscillations of cytoplasmic [ ca ] , and resistance to apoptosis     . this pathway might potentiate the antiapoptotic effect , investigated in this study , derived from an increase of p27 degradation . the goal of the present study was to elucidate the effects of elf - emfs on the functionality of the 20s proteasome , one of the major systems responsible for protein degradation , mainly involved in the removal of oxidized proteins . our data show that the electromagnetic fields are able to affect the proteasome functionality inducing an increase in its proteolytic activity . the finding that egcg , a natural compound with antioxidant properties , counteracts the elf / emfs related pro - oxidant effects demonstrate that this increase in proteasome activity is due to an increase in intracellular free radicals . the modulation of the proteasome functionality may in turn affect indirectly various cellular pathways , that is , apoptosis in the human colon carcinoma cell line ( caco 2 ) . further studies will be necessary to elucidate different susceptibilities to elf - emfs effects between normal and transformed cells in order to gain insight into the role of elf - emfs in the carcinogenetic process . starting from the potential effect on cell proliferation and differentiation , changes in proteasome functionality , which could be the cause of an altered cell cycle , and the degree of protein oxidation will also be considered among the detrimental effects of electromagnetic fields .
electromagnetic fields are an assessed cause of prolonging free radicals lifespan . 
 this study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20s proteasome functionality , the complex responsible for the degradation of oxidized proteins . 
 caco 2 cells were exposed , for 2472 hours , to 1 mt , 50 hz electromagnetic fields . 
 the treatment induced a time - dependent increase both in cell growth and in protein oxidation , more evident in the presence of tpa , while no changes in cell viability were detected . exposing the cells to 50 hz electromagnetic fields caused a global activation of the 20s 
 proteasome catalytic components , particularly evident at 72 hours exposure and in the presence of tpa . 
 the finding that egcg , a natural antioxidant compound , counteracted the field - related pro - oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals .