over the past decade the ability to engineer and produce materials at the nano- or near - atomic scale has triggered rapid product development due to their new interesting properties that were not previously seen at scales above the micrometer .
industrial applications using nanoparticles have resulted in an almost exponentially growing demand for nanosized materials . due to increasing use of nanoparticles in variety of consumer goods , humans are constantly exposed to such nanomaterials besides exposure at production sites [ 15 ] .
unintended exposure to nanomaterials may occur via inhalation , dermal exposure , or gastrointestinal tract absorption and may pose a great risk [ 6 , 7 ] . despite their wide application , little
zinc oxide ( zno ) is among the most commonly utilized group of nanomaterials and has wide - ranging applications . as a well - known photocatalyst ,
zno has received much attention in the degradation and complete mineralization of environmental pollutants [ 9 , 10 ] .
zno nanoparticles ( zno - nps ) are used in industrial products including cosmetics , paints , and medical materials .
zno - nps have external uses as antibacterial agents in ointments , lotions , mouthwashes , and surface coatings to prevent microorganism growth .
zno - nps have also been used as a dietary supplement in human and livestock because zinc can stimulate the immune system and act in an anti - inflammatory way [ 12 , 13 ] .  
many in vitro studies demonstrated that zno - nps are toxic to mammalian cells and are even more toxic than other nanoscale structures of metallic oxide [ 1416 ] . in combination with uv exposure , zno - nps
are known to generate reactive oxygen species ( ros ) like hydroxyl radicals or hydrogen peroxide in aqueous solutions leading to efficient decomposition of organic compounds .
brunner et al .   showed that a three - day exposure of human mesothelioma and rodent fibroblast cell to zno - nps ( 19  nm ) caused dna and mitochondrial damages .  
the human lung is a vulnerable organ for nanoparticle invasion as there is approximately 2300  km of airways and 300 million alveoli , giving rise to a large surface area , which is in contact with the environmental atmosphere and the ultrafine particulate pollutants present in it [ 19 , 20 ] .
adverse systemic reactions have been observed after inhalation of zno fumes or accidental ingestion of large amounts of the metal [ 2123 ] .
inhalation of zno has been found to compromise pulmonary function in pigs and caused pulmonary impairment and metal fume fever in humans [ 24 , 25 ] .
karlsson et al .   found that zno - nps ( 71  nm ) decreased cell viability and caused oxidative dna damage in human alveolar carcinoma epithelial cells ( a549 ) . despite the wide inhalational exposure of zno - nps ,
there are very few studies to demonstrate its toxicity on normal human lung cells and the possible mechanism of toxicity .
therefore , the present study evaluated the toxicity of 50  nm zno - nps in human lung epithelial cells ( l-132 ) and tried to elucidate involvement of oxidative stress therein . as zinc - mediated metallothionein
( mt ) upregulation has been found to protect against oxidative stress - induced cellular injury , attempts were also made to study the effect of zno - nps on expression of mt in l-132 cells .
dulbecco 's modified eagle 's medium ( dmem ) , penicillin , streptomycin , fetal bovine serum ( fbs ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , 2,7-dichlorofluorescin diacetate ( dcfh - da ) , metaphosphoric acid ( mpa ) , dimethyl sulfoxide ( dmso ) , o - phthaldialdehyde ( opt ) , and 5,5-dithiobis(2-nitrobenzoic acid ) were purchased from sigma chemical company ( st .
the zno - nps ( 50  nm ) used in this study were synthesized in drde ( defence research and development establishment , gwalior , india ) , by sol gel method .  
the human lung epithelial cell line ( l-132 ) was purchased from national centre of cell science ( nccs , pune , india ) .
cells were cultured in dmem medium containing 10% fbs and 100  u / ml penicillin - streptomycin - amphotericin mixture and incubated at 37c with 5% co2 .
physicochemical properties of particles were analyzed using transmission electron microscopy ( tem ) , dynamic light scattering ( dls ) , and zeta potential analyzer .
the morphology and size of nanoparticles in the stock dispersion were determined by transmission electron microscopy ( tem ) .
dry powder of particles was suspended in cell culture medium at a concentration of 1  mg / ml and then sonicated at room temperature for 10  min at 40  w to form a homogeneous suspension .
after sonication and stabilization , the tem samples were prepared by drop coating of the stock suspension on carbon - coated copper grids .
tem measurements were performed at an accelerating voltage of 120  kv ( model 1200ex , jeol ltd . , tokyo , japan ) .
zetapals ( brookhaven instruments corporation , holtsville , ny ) was used to determine the hydrodynamic size and zeta potential of particle suspension in cell culture medium .
the sonicated dispersion of zno - nps was used to expose the cells at 5 , 25 , 50 , and 100  g / ml for 24  h. based on the results of previous study done ( data not shown ) with different doses of nanoparticles , a dose range was selected which is also studied by ahamed et al . .
exposure of cells was performed with 80% confluence of cell in 25  cm flasks and 24-well plates in a humidified atmosphere at 37c and 5% co2 .
cells free of zno - nps were used as control cells throughout each assay .  
l-132 cells were plated into a 24-well plate at a density of 50,000 cells / well .
cells were grown overnight in the full medium followed by exposure to zno - np . following the exposure of 24  h ,
the cells were incubated with mtt ( 5  mg / ml ) for 4  h. the medium was then removed , and 200  l of dmso was added into each well to dissolve formazan crystals , the metabolite of mtt . after thorough mixing , the plate was read at 570  nm for optical density that directly correlates with cell quantity .
survival rate was calculated from the relative absorbance at 570  nm and expressed as the percentage of control .  
cells were plated into a 6-well culture plate at a density of 75000 cells / well ( in 2  ml growth medium ) . after overnight growth , supernatants from the culture plates were aspirated out , and fresh aliquots of growth medium containing zno - nps in desired concentrations ( 5100  g / ml ) were added .
upon incubation , cells were washed with phosphate buffered saline ( 0.1  m pbs , ph 7.4 ) , and the morphological changes were observed using leishman 's stain under an inverted phase contrast microscope at 200x magnification .
the production of intracellular reactive oxygen species ( ros ) was measured using dcfh - da .
dcfh - da passively enters the cell , where it reacts with ros to form a highly fluorescent compound , dichlorofluorescein ( dcf ) .
l-132 cells were plated into a 24-well plate at a density of 50,000 cells / well .
the cells were incubated at 37c for 30  min with dcfh - da working solution ( 100  m in methanol ) to yield a 10  m into each well . then cells were washed twice with pbs and exposed to different concentrations of zno - np for 6 , 12 , and 24  h. after exposure to zno - np , the fluorescence was observed at 485  nm excitation and 525  nm emission using a bio - tek synergy ht - i plate reader ( bio - tek instruments , usa ) .
the method is based on a reaction between gsh and o - phthaldialdehyde ( opt ) which gives the fluorescence .
thus gsh concentration in a sample solution can be determined by observing the fluorescence at 360  nm excitation and 420  nm emissions .
l-132 cells were seeded into a 25  cm flask at a density of 1.0  10 cells .
after 24  h exposure to zno - nps , the cells were scraped and pelleted by centrifugation at 5000  rpm for 5  min , then washed in pbs .
the cells were homogenized in 200  l of phosphate - edta buffer ph 8.0 and 80  l of 20% metaphosphoric acid .
the cell homogenate was centrifuged at 16000  rpm at 4c for 30  min .
the assay was performed by taking 100  l supernatants and mixing it with 800  l of phosphate - edta buffer containing 100  l opt ( 10  mg / ml in methanol ) .
after thorough mixing and incubation at room temperature for 15  min , fluorescence was measured at 360  nm excitation and 420  nm emission using bio - tek synergy ht - i plate reader ( bio - tek instruments , usa ) .
results were calculated as nmol of glutathione per mg of protein and presented as a percentage of the control group .
protein assays in the cell lysate were performed using a bca protein assay kit ( pierce , usa ) .
l-132 cells ( 5  10 cells ) exposed to zno - nps ( 5 , 25 , 50 , and 100  g / ml ) for 48  h were collected into tubes and washed with pbs .
the cells were incubated for 3  h in lysis buffer ( 20  mm tris - hcl , ph 8.0 , 5  mm edta , 0.1  m nacl , 0.5% sds , and 100  g / ml rnase ) at 37c . after incubation , phenol  :  chloroform (
1  :  1 ) mixture was used to extract dna . by adding an equal volume of ice - cold absolute isopropanol , dna was precipitated .
dna was dissolved in 50  l of 1x te ( 10  mm tris , 1  mm edta , ph 8.0 ) .
twenty  g of dna was loaded onto 1.5% agarose gel ; electrophoresis was carried out at 60  v for 2  h with tbe as the running buffer .
the fluorescent probe hoechst 33342 stains nuclei specifically , with little or no cytoplasm labeling .
cells exposed to different concentrations of zno - nps ( 50  nm ) were collected and sequentially washed by pbs .
then , the cells were kept in 1  g / ml hoechst working solution for 15  min in the dark at room temperature .
finally , the cells were washed twice with pbs to remove excess stain and examined under fluorescent microscope ( leica - dmlb , germany ) with excitation wavelength of 403  nm .  
human metallothionein ( mt ) messenger rna ( mrna ) expression was determined by reverse transcriptase polymerase chain reaction ( rt - pcr ) .
cells were exposed to 5 , 25 , 50 , and 100  g / ml of zno - np for 24  h , and total rna was isolated using rneasy mini kit ( qiagen , usa ) .
the concentration and integrity of rna were measured using nanodrop spectrophotometer prior to the experiment .
the enhanced avian hs rt - pcr kit ( sigma , usa ) was used for the amplification of human mt-1 and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) gene , according to the manufacturer 's instructions .
the sequences of primer sets used were mt-1 forward , 5-ccactgcttcttcgcttctc-3 and reverse , 5-aggagcagcagctcttcttg-3 ; gapdh forward , 5-ggcgctgagtacgtcgtggagt-3 and reverse 5-cgcctgcttcaccaccttcttg-3. all data were reported as mean  standard deviation ( sd ) .
statistical analysis was performed for the experiments conducted in at least triplicate using one - way anova followed by dunnett test . results with p
other physicochemical characteristics are detailed in table 1 .   mtt assay showed that exposure of human lung epithelial cells ( l-132 ) to 50  nm zno - nps at concentrations of 25 , 50 , and 100  g / ml for 24  h significantly reduced the cell viability in a concentration - dependent manner . however , reduction in cell viability at lower concentration of 5  g / ml was not significant .
as the concentrations of zno - nps increased from 25 to 100  g / ml , the cell viability significantly decreased from 55% to 25% ( figure 1 ) .
the morphology of human lung epithelial cells was examined after zno - nps exposure using phase contrast microscopy .
cells exposed to 50  nm zno - nps for 24  h showed a round shape morphology than control cells ( figure 2 ) . at higher concentration cells appeared to be strongly damaged with a shrunken morphology and detached from the substrate indicating an almost complete destruction of the cells exposed to zno - nps ( figure 2 ) .  
the ability of 50  nm zno - np to induce intracellular oxidant production in l-132 cells was assessed by measuring dcf fluorescence as a marker of ros generation . with the increase of exposure time , a significant ros formation was observed .
zno - nps significantly induced the formation of ros from 16% to 25% at concentration of 25  g / ml to 100  g / ml after 24  h of exposure ( figure 3 ) .  
the intracellular gsh measurement has shown decrease in the intracellular gsh level with the increasing concentrations of zno - nps after 24  h exposure . the significant difference in intracellular gsh level
was observed at 50 and 100  g / ml with remaining gsh of about 14% and 4% , respectively , compared to control ( figure 4 ) .
l-132 cell exposed to 50  nm zno - nps for 48  h showed dna damage ( figure 5 ) , while 24  h of exposure did not show any damage .
extended exposure of zno - nps caused concentration - dependant dna damage which was observed in the form of ladder .
the nuclei of exposed cells appeared to be fragmented , smaller , and rougher , with condensed nuclear material ( figure 6 ) .
metallothionein is known to facilitate metal detoxification and is involved in scavenging of free radicals .
after 24  h exposures of l-132 cells to 50  nm zno - nps , upregulation in mt gene expression was observed .
although the expression was increased initially from concentration of 5 to 50  g / ml , later it decreased at 100  g / ml ( figure 7 ) .
although the beneficial effects of zno - nps have attracted considerable attention in terms of nanomedicine [ 29 , 3638 ] , potential biological and environmental hazards should also be taken into account . in the present study , 50  nm zno - nps exposure significantly reduced cell viability of human lung cells starting at approximately 25  g / ml concentration .
the lowering of cell density and the rounding of cells observed suggest that 25 , 50 , and 100  g / ml nanoparticle concentrations induced substantial cell death .
the cytotoxicity results are in accordance with previous studies [ 16 , 3941 ] .  
oxidative stress as a common mechanism for cell damage induced by nanoparticles is well documented . a wide range of nanomaterial species
have been shown to generate ros both in vivo and in vitro [ 2 , 4244 ] .
similarly , in the present study , 50  nm zno - nps showed generation of ros with significant depletion of reduced glutathione store .
it is well - known that oxidative stress leads to cell death , either by apoptosis or necrosis depending on its extent of severity .
severe oxidative stress to cells causes necrosis while the moderate one causes apoptosis . in the present study , the type of cell death ( apoptosis / necrosis ) after 50  nm zno - nps exposures was evaluated by dna damage analysis and hoechst staining .
formation of the larger dna fragments has been shown to occur in the absence of oligonucleosome formation . moreover ,
cleavage of dna into the larger fragments is sufficient to allow chromatin condensation and subsequent apoptosis in the absence of oligonucleosome formation . in agreement to this
, exposure of 50  nm zno - nps produced large fragments of dna in the range of 100 to 850  bp showing ladder formation in agarose gel .
hoechst staining also showed chromatin condensation and apoptotic body formation at higher concentration of zno - nps , suggesting apoptotic type of cell death .
thus , it is possible that the zno - nps might have induced apoptosis in human lung cells .   since metallothionein ( mt ) is considered as one of the essential biomarkers in metal - induced toxicity   facilitating metal detoxification and protection from free radicals , the ability of zno - nps to modulate the expression of mt gene was also assessed . in present study ,
50  nm zno - np exposed cells showed upregulation of the mt gene . at lower concentrations of zno - nps ,
mt gene expression study indicates that , during the cellular stress - induced by 50  nm zno - nps , initially cells were able to protect themselves by upregulating mt gene using its antioxidant and metal detoxifying properties but at higher concentrations of zno - nps ; same phenomenon did not work because of greater extent of cell stress showing decrease in mt expression . in summary ,
50  nm zno - nps induced cytotoxicity in cultured human lung epithelial cells ( l-132 ) by elevating oxidative stress in a concentration - dependant fashion .
zno - nps also induced dna damage characterized by chromatin condensation and dna ladder pattern illustrating apoptotic type of cell death .
therefore , oxidative stress - induced apoptosis can be considered as one of the pathways of toxicity by zno - nps .
hence , care has to be taken while processing and formulating the nanoparticles till its final finished product .
 zinc oxide nanoparticles ( zno - nps ) are increasingly used in sunscreens , biosensors , food additives , pigments , rubber manufacture , and electronic materials . with the wide application of zno - nps 
 , concern has been raised about its unintentional health and environmental impacts . 
 this study investigates the toxic effects of zno - nps in human lung cells . in order to assess toxicity , human lung epithelial cells ( l-132 ) were exposed to dispersion of 50  nm zno - nps at concentrations of 5 , 25 , 50 , and 100  g / ml for 24  h. the toxicity was evaluated by observing changes in cell morphology , cell viability , oxidative stress parameters , dna damage analysis , and gene expression . 
 exposure to 50  nm zno - nps at concentrations between 5 and 100  g / ml decreased cell viability in a concentration - dependent manner . 
 morphological examination revealed cell shrinkage , nuclear condensation , and formation of apoptotic bodies . 
 the oxidative stress parameters revealed significant depletion of gsh level and increase in ros levels suggesting generation of oxidative stress . 
 zno - nps exposure caused dna fragmentation demonstrating apoptotic type of cell death . 
 zno - nps increased the expression of metallothionein gene , which is considered as a biomarker in metal - induced toxicity . 
 to summarize , zno - nps cause toxicity in human lung cells possibly through oxidative stress - induced apoptosis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion