palladium ( pd ) is a component of the catalytic converters that promotes reduction and oxidation of pollutants in exhaust gases to less harmful ones . catalytic converters emit pd mainly as particulate matter in the breathable size range ( d < 2.5  m ) [ 2 , 3 ] , containing also pd - np ( d < 100  nm ) . indeed , after the introduction of catalytic converters based on the pt group elements , the level of pd ( also in the form of nanoparticles ) rapidly increased in the areas bordering roads [ 510 ] . at the same time , in europe , allergic sensitization to pd increased [ 1114 ] . a causative role for pd(ii ) ions in the development of allergic symptoms has been deduced from studies on occupationally exposed people [ 1416 ] and people wearing pd - containing dental alloys   and is sustained by laboratory data . pd being chemically related to nickel , one of the most important sensitizing metals , phenomena of cross - reactivity might contribute to the increased frequency of immune reactions to pd [ 1921 ] . few data are available to explain the role of pd - np for the allergic sensitization . our recent data confirm the sensitizing power of pd(ii ) and pd(iv ) ions [ 22 , 23 ] . moreover , we observed that pd - np can induce secretion of inf- , a th1 cytokine involved in type iv immune reactions   suggesting a role for polluting pd - np in the raised frequency of allergic contact dermatitis to pd in people living in urban settings . on the other hand , no data are available for the possible involvement of pd in cancer development , which is one of the major concerns about metal - based nps . in the ionic form , pd(ii ) does not induce significant genotoxicity , but no extrapolations are possible to pd - np . in fact , nps have peculiar physical properties that may not be simply predicted by data from the corresponding ions . recently , our group found that highly cytotoxic co ions are released from co - nps , but only nanoparticles are rapidly accumulated within the nucleus and interact with dna , induce transformation , and affect molecular pathways implicated in carcinogenesis and inflammation . all these findings were obtained at rather high exposure concentrations . in the present work , we have studied the effects of low , subtoxic pd - np doses . as a target , we used cells of the immune system , taking into account that palladium can act as hapten able to induce an immune reaction . in particular , to study the possible interference of pd - np in the cell cycle progression , we have exposed to pd - np normal human pbmcs entering the first in vitro mitotic division . zerovalent pd - np ( 28  nm diameter ) were produced and characterized as described previously . the pd - np stock solution did not contain pd ions , even after long time from preparation , as assessed by low resolution inductively coupled plasma mass spectrometry . to exclude the presence of agglomerates , the pd - np stock solution was treated by ultrasonic bath for 10  min at 100  hz ( elmasonic s , elma , singen , germany ) and then immediately diluted in complete culture medium at working concentrations and given to the cells straight away . potassium hexachloropalladate ( k2pdcl6 , sigma aldrich , milan , italy ) ( pd(iv ) ion ) was used at subtoxic concentration , for comparison . human peripheral blood mononuclear cells ( pbmcs ) from 3 healthy donors were isolated by density gradient centrifugation using a commercial separation medium consisting of a mixture of ficoll 400 and sodium diatrizoate ( lymphoprep , stemcell technologies , voden medical instruments , milan , italy ) , according to standard manufacturer procedure . cells were seeded at 500,000 cells ml in rpmi-1640 medium supplemented with 10% fetal calf serum ( complete culture medium ) containing 5  g  ml phytohemagglutinin - l ( pha - l ) ( sigma aldrich , milan , italy ) , an initiator of mitosis of normal human leukocytes . to evaluate the effect of the mitogen on cell viability and number , the pha - stimulated cells were examined every 24  h for 5 days by trypan blue exclusion using a neubauer counting chamber as described . after 48 hours of culture in the presence of pha , pd - np ( or pd(iv ) ions or an equal volume of the vehicle , as controls ) were added to the cultures for further 48 hours and finally harvested appropriately for analysis . cytotoxicity was assessed by mtt ( 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide ) test according to the standard procedure . briefly , pbmcs ( 40,000 per well in 200  l culture medium , four replicates ) were seeded in a 96-well culture plate , incubated at 37c , 5% co2 , and exposed to increasing concentrations of pd - np or pd(iv ) ion ( 0.1 , 1 , 5 , 10 , 20 , 40 , 80  g  ml ) or to an equal volume of vehicle ( rpmi , 10% fcs ) to assess 100% viability . at the end of the exposure time , a washing step was performed to remove the incubation medium containing unreacted mtt and pd - np or pd(iv ) ion . then , 20  l of the mtt solution ( 5  mg  ml ) was added to each well and incubated for an additional 3 hours . the colored formazan crystals produced from mtt reduction were dissolved in 200  l of dimethyl sulfoxide ( dmso ) . the optical density ( o.d . ) values of the solutions were measured at 540  nm as excitation wavelength using a spectrophotometer plate reader ( applied biosystems , life technologies , monza , italy ) . also , negative controls ( i.e. , complete culture medium alone or complete culture medium containing pd - np ) have been tested and no interference with the colorimetric assay was observed , at all concentrations used . cells without any treatment were assumed as positive control ( 100% viability ) . the highest concentration that , after 48  h incubation , caused signal reduction ( cell death ) by less than 10% compared to the control was used for the experiments . the production of intracellular reactive oxygen species ( ros ) was indirectly measured using dichlorodihydrofluorescein diacetate ( dcfh - da ) , a nonfluorescent and cell - permeant substance converted by endogenous esterases to dichlorofluorescein ( dcfh ) , and nonfluorescent nonpermeant compound that cellular ros oxidize proportionally to fluorescent dichlorofluorescein ( dcf ) . after 48 hours , cells were washed with phosphate - buffered saline ( pbs ) and incubated with 10 m dcfh - da , for 30  min , at 37c . finally , the fluorescence emitted by the oxidized form of dcfh - da was measured using a cytofluor fluorescence multiwell plate reader ( applied biosystems , life technologies , monza , italy ) with the excitation fluorescence wavelength at 485  nm and the emission one at 530  nm . pbmcs were fixed overnight at 4c in 2% paraformaldehyde and 2% glutaraldehyde in pbs , postfixed in 1% osmium tetroxide in veronal acetate buffer ( ph 7.4 ) for 2  h at room temperature , stained with 2% uranyl acetate , dehydrated in acetone , and embedded in epon 812 . thin sections were examined under a philips cm10 tem after poststaining with uranyl acetate and lead hydroxide . cells were fixed in 70% cold ethanol , stained with 50  g / ml propidium iodide in pbs buffer containing 200  g / ml rnase ( dnase - free , sigma aldrich , milan , italy ) , and analyzed using a facscalibur flow cytometer equipped with the cellquest software ( becton dickinson ) . debris were excluded from the analysis after gating them out by setting the forward scatter versus side scatter plot on the viable cells area . cell doublets and aggregates were excluded by gating fl2-area versus fl2-width . the low flow rate mode ( 400500 events sec ) finally , data were analysed using modfit lt software ( verity software house , toshan , me , usa ) . statistical analysis was performed using graphpad prism 5.0 software ( la jolla , ca , usa ) . mtt and ros data were analyzed by student 's t - test ; cell cycle data were analyzed by three- and four - way analysis of variance ( anova ) with tukey multiple comparisons correction , when appropriate . the comparison between the different groups in different concentrations was performed with the t - test . in all cases pbmcs , which are recognized as quiescent cells ( g0 state ) , were induced to enter the cell cycle by in vitro stimulation with pha , a well - known mitosis inducer . after a latency period of 48 hours , pd - np were added 48 hours after pha , at the beginning of the growth phase . after 4 hours of exposure to pd - np , no evident changes in cell viability were observed at any tested dose . prolongation of the exposure time to 24 hours showed a concentration - dependent decrease of viability up to 40  g  ml and death at 80  g  ml . at 48 hours , also 40  g  ml treated cells were incapable of metabolic conversion of mtt , and the same result was obtained after 72 hours of exposure . pd(iv ) ions were more toxic , causing almost complete loss of cell viability earlier , at 48 hours and at lower dose ( 5  g  ml ) . therefore , the following experiments were performed using the nontoxic doses of 10  g  ml pd - np and 0.1  g  ml pd(iv ) ions , for 48 hours . a moderate , but not statistically significant increase of intracellular ros was measured both in controls and in pd - np exposed cells , whereas ros significantly ( p < 0.05 ) increased after pd(iv ) exposure by more than 30% of the control ( figure 2 ) . electron microscopy revealed that , as compared to controls , pbmcs exposed to pd - np and to pd ions shared marked subcellular alterations ( figure 3 ) . most notably , these included the presence of numerous autophagosomal vacuoles containing damaged mitochondria and/or undigested cytoplasmic material , as well as the accumulation of multilamellar bodies and lipid droplets . such alterations were more marked in pbmcs exposed to pd ions , being already well evident at our nontoxic concentration . with both treatments , mitochondria showed evidence of damage , as indicated by condensation and loss or swelling of cristae . furthermore , in both cases , there was evidence of increased plasma membrane ruffling and nuclei were more indented and/or convoluted compared to controls . in pbmcs exposed to pd - np , electron - dense agglomerates of nanosized particulate material within or adjacent to cytoplasmic vesicles were consistent with the internalization of pd - np . the examination of cell cycle ( figure 4 ) confirmed the quiescent / resting state of all unstimulated pbmcs either freshly isolated ( w / o pha , 0 hours ) or cultured for the entire experimental period ( w / o pha , 96 hours ) , as they were found in the g0 state . the addition of the polyclonal activator pha stimulated cell division , as shown by their distribution throughout all phases of the cell cycle ( g0/g1 : 43.1  1.9% ; s : 48.8  2.3% ; g2/m : 8.1  1.2% ) . the exposure of pha - activated pbmcs to 10  g  ml pd - np for the time allowing the completion of at least one cell division ( 48  h ) was associated with a significant reduction of cells synthesizing dna ( s - phase ) ( 35.4  1.5% versus 48.8  2.3% , p < 0.05 ) , a significant increase of cells within the g0/g1-phase ( 59.8  1.8% versus 43.1  1.9% , p < 0.05 ) , and a significant reduction of cells in g2/m - phase ( 4.8  0.1% versus 8.1  1.2% , p < 0.05 ) , compared with unexposed control cells . a different cell cycle outline was observed for pd(iv ) ion exposed cells , with significantly higher g0/g1 accumulation compared to pd - np exposed cells ( 74.9  2.2% versus 59.8  1.8% , p < 0.05 ) , lower percent of cells in s - phase ( 25.1  1.2% versus 35.4  1.5% , p < 0.05 ) , and no cells engaged in the cell division ( g2/m ) . the vehicles alone did not cause significant changes of the cell cycle distribution . also , pbmcs were evaluated for cell cycle distribution after exposure to pd - np and pd(iv ) , in absence of pha stimulation ; in no case were these compounds found to induce cell cycle initiation and all cells remained in the g0 phase ( data not shown ) . sub - g0/g1 cells , indicative of cell death , were largely absent from the analyzed populations , for all tested samples . in the present work , we have found that pd - np exposure is associated with an increased percentage of mitogen - activated pbmcs with a diploid dna content , indicative of maintenance of g0 state or prolongation / arrest in g1-phase . studying the influence of potential toxicants on the g1-phase of the cell cycle is of central importance ; in fact , most , if not all , human cancer types show a deregulated control of g1 progression , a period in which cells decide whether to start proliferation or stay quiescent [ 31 , 32 ] . important processes , such as increase in cell size and centrosome duplication , are initiated in g1 and are thus controlled . control is conducted by checkpoint complexes that stall the cell cycle until the appropriate repair is completed . if such repair is not achieved , the cell may pause or undergo senescence or apoptosis . alteration of repair ( controlled by caretakers ) and checkpoint complexes ( controlled by gatekeepers genes ) is central in oncogenesis . in our experiments , pbmcs , which are mostly quiescent cells in the g0 state and prompted to enter the cell cycle by in vitro stimulation with pha , were exposed to low noncytotoxic dose - time combinations of pd - np and pd ions . under these experimental conditions , pd - np gain entry to the cytosol , where they appear in clusters lacking a membranous envelope , suggesting the possibility to exert a catalysing redox activity   on cellular elements . at the ultrastructural level the alterations observed in the pbmcs exposed to pd - np largely corresponded to those observed in the pbmcs exposed to pd ions . most notably , in both cases the cells showed evidence of mitochondrial damage and of mitophagy , that is , autophagic elimination of damaged mitochondria . mitophagy is a key protective mechanism against mitochondrial damage and the consequent ros - induced cellular alterations . the accumulation of abundant multilamellar bodies is also in agreement with the macroautophagic response , as these structures originate via autophagy and reflect the accumulation of membrane lamellar material selectively resistant to lysosomal degradation within autophagolysosomes . lipid droplets , specialized organelles for the deposition and storage of neutral lipids , are associated with common pathologies linked to lipid accumulation and mitochondrial damage . their accumulation in pbmcs exposed to pd is fully consistent with the loss of mitochondrial fatty acid beta oxidation due to mitochondrial damage , that , in turn , results in lipid accumulation and loss of atp production . in conclusion , the morphological evidence strongly suggests that pd ions , per se or dissolved from nps , accumulate in the mitochondria , as we recently demonstrated in the case of cobalt . here , acting as catalyst , ionic pd may interfere with the oxidative phosphorylation ( oxphos ) . the mitochondria , whose respiratory activity is damaged by the influx of pd ions , will be poor atp producers and will release excessive amounts of ros . the ros - induced cellular stress is predicted to arrest the cell cycle , acting via oxidative stress - sensitive cell cycle regulator genes such as p53 and cdkn2a , which we have recently found to be implicated in the response to co particles in balb/3t3 cells . in turn , ros and atp depletion activate cell death pathways , which induce the release of proapoptotic proteins from mitochondria . macroautophagy may mitigate these effects by the selective sequestration and subsequent degradation of the dysfunctional mitochondria and other ros - damaged subcellular structures before the activation of prodeath pathways [ 30 , 35 , 36 ] . thus , macroautophagy , and , in particular , mitophagy , most likely represents a key prosurvival pathway in cells exposed to palladium . autophagy has been demonstrated to be marker of heavy metals toxicity for human hematopoietic progenitor cells , representing a protecting and recycling catabolic pathway removing damaged organelles and molecules . the cellular outcome of inducing autophagy in response to stress is complex and depends on the cellular context , type , and magnitude of stress . in mild stress conditions , autophagy may enhance cell survival by allowing the cell to engage dna repair mechanisms and checkpoint activation . nontoxic dose of pd ions induced a more marked production of ros relative to pd - np , contrary to almost all other metal - based nps . however , local ros generation at sites of pd - np accumulation , not measurable by the overall method used , might be possible at the nontoxic doses applied in our experiments . in fact , local concentration is important for the cellular function of ros . on the other hand , changes in ros generation may modulate autophagic processes , as ros can modify activating molecules to stimulate or inhibit autophagy and engages in cross - talk with autophagy in both cell signaling and protein damage . mechanisms of cell damage by pd - np can be also explained through cell cycle disturbance by the withdraw / inactivation of nutrients and/or pha or by modulation / inhibition of downstream signaling due to their catalytic activity , as described for polystyrene - coated pd nanoparticles . therefore , pd - np might produce an effect similar to nutrient depletion in inducing autophagy in the g1 phase . it is possible that pd - np interference with mitogen / growth factors would prolong the g1-phase checkpoint called restriction point that prevents cells from entering the cycle until they have accumulated a certain threshold of mitogen - induced events . the described findings suggest a potentially harmful role for low dose pd ions and pd - np in hematopoietic cells leaving quiescence . however , it will be essential to verify whether the observed disturbance of the transition from quiescence to cell division represents a temporary response or might lead to permanent alterations of the cell functions . to this aim , long - term exposure to low dose of pd (
there is concern about the possible toxicity of palladium nanoparticles ( pd - np ) , as they are released in the environment through many applications . 
 we previously studied the toxicity of pd - np at high concentrations ; here we address the possible toxicity of pd - np at low , subtoxic doses . 
 in particular , we have exposed normal human pbmc entering into the first in vitro mitotic division to pd - np and to pd(iv ) ions to evaluate ros generation and cell cycle progression . 
 we have measured a statistically significant increase of intracellular ros in pd(iv ) exposed cells , but not in pd - np exposed cells . 
 tem revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles , which appeared more conspicuous in cells exposed to pd(iv ) ions than to pd - np . 
 pd - np were visible in the cytoplasm of pd - np exposed cells . 
 pd - np addition was associated with a significant increase of cells within the g0/g1-phase and a significant reduction in gs- and g2/m - phases . 
 cells exposed to pd(iv ) ions showed a significant amplification of these cell cycle alterations . 
 these results suggest that ions , per se or released by nps , are the true inducers of pd toxicity 
 . it will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations .