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Toxicology 302 (2012) 203– 211

Contents lists available at SciVerse ScienceDirect

Toxicology

jou rn al hom epage: www.elsev ier .com/ locate / tox ico l

ong-lasting oxidative pulmonary insult in rat after intratracheal instillationf silica nanoparticles doped with cadmium

eresa Coccinia,∗, Elisa Rodaa, Sergio Barnib, Cinzia Signorini c, Luigi Manzoa

Salvatore Maugeri Foundation IRCCS Institute of Pavia, and University of Pavia, Toxicology Division and European Centre for Nanomedicine, Pavia, ItalyUniversity of Pavia, Department of Biology and Biotechnology “L. Spallanzani” Pavia, ItalyUniversity of Siena, Department of Pathophysiology, Experimental Medicine, and Public Health, Siena, Italy

r t i c l e i n f o

rticle history:eceived 17 May 2012eceived in revised form 30 July 2012ccepted 31 July 2012vailable online 9 August 2012

eywords:n vivoanotoxicology

soprostanesuperoxide dismutaseNOSOX

a b s t r a c t

Silica/cadmium containing nanomaterials are now produced on industrial scale due to their potential fora variety of technological applications. Nevertheless, information on toxicity, exposure and health impactof these nanomaterials is still limited. In this study, in vivo effects of silica nanoparticles (SiNPs) dopedwith Cd (SiNPs-Cd, 1 mg/rat), soluble CdCl2 (400 �g/rat), or SiNPs (600 �g/rat) have been investigatedby evaluating F2-isoprostanes (F2-IsoPs), superoxide dismutase (SOD1), inducible nitric oxide synthase(iNOS) and cyclooxygenase type 2 (COX-2) enzymes, as markers of oxidative stress, 24 h, 7 and 30 daysafter intra-tracheal (i.t.) instillation to rats. Free and esterified F2-IsoPs were evaluated in lung and plasmasamples by GC/NICI-MS/MS analysis, and SOD1, iNOS and COX-2 expression in pulmonary tissue byimmunocytochemistry.

Thirty days after exposure, pulmonary total F2-IsoPs were increased by 56% and 43% in CdCl2 andSiNPs-Cd groups, respectively, compared to controls (32.8 ± 7.8 ng/g). Parallel elevation of free F2-IsoPswas observed in plasma samples (by 113% and 95% in CdCl2 and SiNPs-Cd groups, respectively), compared

to controls (28 ± 8 pg/ml). These effects were already detectable at day 7 and lasted until day 30 post-exposure. Pulmonary SOD1-, iNOS-, and COX-2-immunoreactivity was significantly enhanced in a time-dependent manner (7 days <30 days) after both CdCl2 and SiNPs-Cd treatments. SiNPs did not influenceany of the evaluated endpoints.

The results indicate the capacity of engineered SiNPs-Cd to cause long-lasting oxidative tissue injuryfollowing pulmonary exposure in rat.

. Introduction

Silica/cadmium containing nanomaterial application inedicine and technology has attracted much attention in the

atest years (Rzigalinski and Strobl, 2009; Vivero-Escoto et al.,010). However, information on toxicity, exposure and health

mpact is still limited to reach resolute conclusions on the risksosed by these nanomaterials. With the exponentially growingroduction of engineered nanoproducts, occupational respiratoryxposure to cadmium nanoparticles is increasing, and manyspects related to the size of these nanomaterials, smaller than

ells and cellular organelles, have raised concerns about safetyMaynard et al., 2006; Oberdörster et al., 2005a,b). Several reportsave been released on the relationship between oxidative stress

∗ Corresponding author at: Toxicology Division, Salvatore Maugeri Foundationedical Center IRCCS, Via Maugeri, 10, 27100 Pavia, Italy. Tel.: +39 0382 592416;

ax: +39 0382 24605.E-mail address: teresa.coccini@fsm.it (T. Coccini).

300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tox.2012.07.019

© 2012 Elsevier Ireland Ltd. All rights reserved.

and inflammation induced by nanoparticles although their respec-tive and/or causative role in producing inflammatory diseaseprocesses in the lung is still unclear (Di Gioacchino et al., 2011; Liet al., 2008).

Most in vitro studies on silica nanoparticles (SiNPs) reportresults of cellular uptake, size- and dose-dependent cytotoxicity,increased reactive oxygen species levels and pro-inflammatorystimulation (Eom and Choi, 2011; Lee et al., 2011). A limited numberof in vivo studies demonstrate largely reversible lung inflammation,granuloma formation and focal emphysema, with no progressivelung fibrosis after exposure via respiratory tract (Napierska et al.,2010).

On the other hand, Cd per se is toxic to the respiratory tractand other organ systems when inhaled (Oberdörster et al., 1994;Potts et al., 2001). Inhalation of Cd can result in acute insult such asoedema, or in chronic injuries like emphysema, pulmonary fibrosis

or adenocarcinomas. Although the mechanisms of Cd toxicity arenot yet fully understood, several investigations reported evidencefor interference with essential metals, induction of oxidative stress,disruption of cadherins, inhibition of DNA repair, interference with

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Plasma free, and lung total F2-IsoPs were expressed as picograms or nanograms

04 T. Coccini et al. / Toxic

poptosis (Bertin and Averbeck, 2006), and induction of pulmonarynflammatory response.

Health effects of nanoparticles manufactured from metals, as inhe case of SiNPs-Cd, may be not necessarily easily predicted fromrevious studies of non-nanoparticulate quantities of the sameetals.In this study, we examined the in vivo effects of silica nanopar-

icles (SiNPs) doped with cadmium (SiNPs-Cd) compared to thoseaused by the soluble CdCl2 in terms of their capacity to gener-te free radicals and induce oxidative stress, and proinflammatoryffects in lung tissue of rats intratracheally (i.t.) treated. TheiNPs-Cd response was assessed by measuring lung and plasmaevels of F2-isoprostanes, as known markers of oxidative stressMilatovic et al., 2011), and immunocytochemical analysis of pul-

onary copper–zinc superoxide dismutase (Cu/Zn-SOD1 or SOD1),n antioxidant isozyme also involved in oxidative stress pathway.he expression of two inducible enzymes, namely nitric oxide syn-hase (iNOS) and cyclooxygenase type 2 (COX-2), which synthesizeitric oxide (NO) and prostaglandin E2 (PGE2), respectively undertimulation by oxidative stressors including cadmium (Moralest al., 2006), were also evaluated by immunocytochemistry in pul-onary tissues. The effect of SiNPs-Cd was assessed at 24 h, 7 and

0 days post-exposure in comparison with that caused by admin-stration of equivalent amounts of CdCl2 and SiNPs.

. Materials and methods

.1. Synthesis and characterization of nanoparticles

SiNPs-Cd were produced by impregnation of SiO2 (silica nanosize HiSilTM T700ommercial Degussa, average pore size 20 nm, surface area 240 m2/g). Impreg-ation of SiO2 with cadmium nitrate dehydrate was obtained by mixing CdNO3

.56 × 10−2 M in an aqueous solution with silica dispersed in a concentration ratioeading to a sample containing 40% Cd by weight. Dispersion was brought to drynessnd treated at 600 ◦C for 2 h in air in order to remove the residual organic groups.his heat treatment leads to the decomposition of nitrate Cd to Cd oxide, dispersedn silica. Powder was later subjected to grinding mills with high energy (200 rpm for.5 h, 400 rpm for 1.5 h, 600 rpm for 2 h) to get the most equal distribution of parti-le size and/or aggregates of particles. The thermal processes have also allowed toemove possible endotoxin contamination (e.g. lypopolysaccharides) according tohe Food and Drug Administration guidelines (Vallhov et al., 2006).

Attention was devoted to tune the thermal treatments in order to retain therains dimensions, pores size, and distributions, and particles surface areas. Thentire synthesis preparation was performed under sterile condition (e.g. usingaminar flow cabinet, ultrapure water, autoclaved glassware) to avoid NPs con-amination. An endotoxin assay confirms the absence of detectable gram negativendotoxin on both NP types, namely SiNPs and SiNPs-Cd, at the concentration of

mg/ml (the detection limit was less than 0.1 EU/ml).Physicochemical characterization of SiNPs-Cd was performed by STEM (scan-

ing transmission electron microscopy) technique, X-ray powder (XRD) diffraction,nd dynamic light scattering (DLS) as previously described (Coccini et al., in press).riefly, STEM quantitative analyses showed the aggregation of SiNPs-Cd and thenalysis of the elements in HAADF mode (EDS spectra) confirmed the presence ofd, Si and O. X-ray diffraction demonstrated amorphous and crystalline phases ofhe sample. Dynamic light scattering determination of the SiNPs-Cd size distribu-ion showed tendency to form aggregates and agglomerates of about 350 nm, and

zeta potential of −23 mV (in deionized water). Although particle samples wereortexed just before measurement, tendency to agglomerate was detected. Parti-les presented spherical form, primary particle size range of 20–80 nm and specificurface area of about 200 m2/g. Metal impurities and the release of cadmium fromanoparticles dispersed in physiological solution were determined by flame-atomicbsorption analysis.

Maximum cadmium release was 15% after 16 h in physiological solution. Fur-her metal release was negligible in the subsequent 10-day period. Cadmium andilica contents in the nanoparticles were 32.5% and 24.1%, respectively. The mainmpurities were Ca (0.3%), Na (0.2%), K (0.2%), Fe (0.04%) and Mn (0.001%). Other

etals were present in quantities less than 1‰.

.2. Animal treatment

Adult male Sprague–Dawley rats (12 weeks old), purchased from Charles Rivertalia (Calco, Italy), were allowed to acclimatize for at least 2 weeks before treat-

ent, and kept in an artificial 12 h light:12 h dark cycle with humidity at 50 ± 10%hroughout the experiment. Animals were provided rat chow (4RF21 diet) and tapater ad libitum.

302 (2012) 203– 211

Rats were treated with a single intratracheal (i.t.) instillation of SiNPs-Cd(1 mg/rat, corresponding to about 250 �g Cd/rat). Separate groups of animalsreceived by i.t.: an equivalent cadmium dose as CdCl2 (400 �g Cd/rat), SiNPs(600 �g/rat) or 0.1 ml saline/rat (control). In previous studies (Damiano et al., 1990;Driscoll et al., 1992), a single intratracheally instilled dose of 400 �g CdCl2/rat wasshown to induce in rats moderate lung injury evolving into chronic inflammationand fibrosis.

All experimental procedures were performed in compliance with the EuropeanCouncil Directive 86/609/EEC on the care and use of laboratory animals. The weightof rats in all treatment groups at the time point 0 (day of instillation) was g 278 ± 3.

Groups of rats (n = 6 for each treatment group at each time point) were anes-thetized with pentobarbital sodium and i.t. instilled with the test materials. SiNPs-Cdsuspension was vortexed on ice just before the exposure to force nanoparticle dis-persion and avoid agglomerate formation. No surfactants or solvents were used.

All endpoint evaluations were performed 24 h, 7 and 30 days after treatment:at each time point, rats were deeply anesthetized by an overdose i.p. injection of35% chloral hydrate (100 �l/100 g b.w.). From a set of animals (n = 3), lungs wereremoved and blood collected in heparinized tubes for the isoprostane analyses. Theblood samples were centrifuged at 2400 × g for 15 min at 4 ◦C; the platelet-poorplasma was saved and the buffy coat was removed by aspiration.

From the remaining set of animals (n = 3), lung preparation for morphohisto-chemical evaluations was done by vascular perfusion of fixative. Briefly, the tracheawas cannulated, and laparotomy was performed. The pulmonary artery was can-nulated via the ventricle, and an outflow cannula was inserted into the left atrium.In quick succession, the tracheal cannula was connected to about 7 cm H2O pres-sure source to inflate the lungs with air, and clearing solution (saline with 100 U/mlheparin, 350 mosM sucrose) was perfused via the pulmonary artery. After bloodwas cleared from the lungs, the perfusate was switched to fixative consisting of 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, the lungs werecarefully removed.

2.3. Oxidative stress and pro-inflammatory evaluation

2.3.1. F2-isoprostanes (F2-IsoPs) in lung and plasmaF2-IsoPs, a series of prostaglandin F2-like compounds, are formed by free

radical-catalyzed peroxidation of phospholipid-bound arachidonic acid, througha cyclooxygenase-independent pathway. Initially formed in situ on phospho-lipids (esterified F2-IsoPs), are released into the circulation (free F2-IsoPs), andowing to their relatively low reactivity, F2-IsoPs can be easily measured inbiological samples. As a consequence, F2-IsoPs are considered specific and reli-able oxidative stress markers in vivo (Milne et al., 2005; Morrow & Roberts,1997).

Plasma and lung samples were examined for free and total (sum of free and ester-ified) F2-IsoPs, respectively, as indicators of free radical-induced lipid peroxidation.The determinations of the F2-IsoPs were performed by gas chromatography/tandemmass spectrometry (GC/NICI-MS/MS) analysis, which has proved to be applicable instudies in vitro and in vivo (Aldinucci et al., 2007; De Felice et al., 2009; Signoriniet al., 2003, 2008, 2009) with good reliability (in term of specificity, repeatabilityand accuracy), whereas the results of some E.L.I.S.A. immunoassay methods havebeen repeatedly questioned (Bessard et al., 2001; Il’yasova et al., 2004; Proudfootet al., 1999; Soffler et al., 2010).

For plasma free F2-IsoPs determination, butylated hydroxytoluene (BHT)(90 �M) was added to the plasma as an antioxidant. An aliquot of plasma (1 ml)was spiked with tetradeuterated prostaglandin F2� (PGF2�-d4) (500 pg in 50 �l ofethanol; Cayman, Ann Arbor, MI, USA), as an internal standard. After acidification,by the addition of 2 ml of acidified water (pH 3), the extraction and purificationprocedure were carried out. It consisted of two solid-phase separation steps: anoctadecylsilane (C18) cartridge followed by an aminopropyl (NH2) cartridge (Waters,Milford, MA, USA) (Signorini et al., 2003).

For total F2-IsoPs, the lung tissue was homogenized (10% w/v) in phosphate-buffered saline (PBS), pH 7.4. and 100 �M BHT was added. Five hundred microlitresof 1 M KOH were added to the lung homogenate (1 ml) and the sample was incubatedat 45 ◦C for 45 min. Subsequently, the pH was adjusted to 3.0 by adding HCl (1 M,500 �l). The sample was then spiked with PGF2�-d4. Total lipids were extracted byaddition of ethyl acetate (10 ml). The total lipid extract was applied onto an NH2

cartridge and isoprostanes eluted (Signorini et al., 2009).A derivatization step followed the solid-phase extraction. The carboxylic group

was derivatized as the pentafluorobenzil ester whereas the hydroxyl groups wereconverted to trimethylsilyl ethers. In the GC/NICI-MS/MS analysis, the measuredions were the product ions at m/z 299 and m/z 303 derived from the [M−181]− pre-cursor ions (m/z 569 and m/z 573) produced from 15-F2t-IsoPs, the most representedF2-IsoP isomer, and the tetradeuterated derivative of the PGF2�-d4, respectively(Signorini et al., 2003).

per millilitre, respectively. The calibration curve correlations were adequate(r2 = 0.994 for free F2-IsoPs; and r2 = 0.9987 for total F2-IsoPs); accuracy was 97.8%(free F2-IsoPs), and 98.5% (total F2-IsoPs); variability coefficient were 2.5, and2.2%, for free and total F2-IsoPs, respectively. The minimum detection limit was5 pg/ml.

ology 302 (2012) 203– 211 205

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Fig. 1. Total F2-Isoprostane levels in lungs. Pulmonary F2-IsoPs levels have beenevaluated at 24 h, 7 and 30 days after i.t. treatment with CdCl2, SiNPs, and SiNPs-Cd.Data are means ± standard deviation (SD). Differences between groups were evalu-ated using two-tailed t test and p value < 0.05 was considered statistically significant

T. Coccini et al. / Toxic

.3.2. SOD1, iNOS and COX-2 in lung

.3.2.1. Lung sampling. The top and the bottom regions of the right lungs of con-rol and differently treated animals were dissected. Tissue samples were obtainedccording to a stratified random sampling scheme which is a suggested methodor lung tissue in order to compensate for regional differences, known to exist inung (Weibel, 1979), and to reduce the variability of the sampling means. Then,hese samples were washed in NaCl 0.9% and post-fixed by immersion for 7 h in 4%araformaldehyde in 0.1 M phosphate buffer (pH 7.4), dehydrated through a gradederies of ethanol and finally embedded in Paraplast. Eight-micrometre thick sectionsere cut in the transversal plane and collected on silan-coated slides.

.3.2.2. Immunocytochemistry for SOD1, iNOS, COX-2. The reactions were carried outimultaneously on slides of control and treated animals at all stages, in order to avoidossible staining differences due to small changes in the procedures.

Immunocytochemistry was performed, using commercial antibodies on rat lungpecimens, to localize presence and distribution of three different markers indicativef inflammation-related oxidative stress: (i) Cu–Zn superoxide dismutase-1 (SOD1),ii) inducible nitric oxide synthase (iNOS) and (iii) cyclooxygenase-2 (COX-2).

Lung sections of control and treated rats were incubated overnight at roomemperature with: (i) primary rabbit polyclonal antibodies against SOD1 or iNOSSanta Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100 or (ii) a primary goatolyclonal antibody against COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)iluted 1:100; all the employed antibodies were diluted in PBS.

Biotinylated anti-rabbit, and anti-goat secondary antibodies and an avidiniotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame,A, USA) were used to reveal the sites of antigen/antibody interaction. The 3,3′-iaminobenzidine tetrahydrochloride peroxidase substrate (Sigma, St. Louis, MO,SA) was used as chromogen, and Haematoxylin was employed for nuclear coun-

erstaining. Then, the sections were dehydrated in ethanol, cleared in xylene, andnally mounted in Eukitt (Kindler, Freiburg, Germany).

As negative controls, some sections were incubated with phosphate-bufferaline in absence of the primary antibodies; no immunoreactivity was observedn this condition.

.3.2.3. Grading of labelling. A scoring system was used to evaluate the degreef immunostaining for SOD1, iNOS, and COX-2 using conventional brightfieldicroscopy according to a semiquantitative scale ranging from undetectable

−) to strong (+ + + +). The localization and intensity of labelling was recordednd graded as follows, with approximate percentages indicating those numbersf relevant cells showing a intense positive reaction: (−) absent/undetectablemmunohistochemical reaction; (+) mild positivity involving 1–10% of organ cells;++) moderate immunoreactivity involving up to approximately 25% cells; (+++)trong immunopositivity involving up to approximately 50% cells; (++++) maximalmmunohistochemical reaction involving more than approximately 50%.

The slides were observed and scored with a bright-field Zeiss Axioscop Plusicroscope. The images were recorded with an Olympus Camedia C-2000 Z digital

amera and stored on a PC running Olympus software.

.3.3. Lung histology and ultrastructural morphologyFrom the second set of rats (n = 3 for each treatment at each time point), pul-

onary tissues were also processed for the following morphological evaluations.

.3.3.1. Histology. Lung sections of control and treated rats were stained withaematoxylin/Eosin (H&E) to evaluate overall tissue structural changes.

.3.3.2. Electron microscopy. Lung fragments (small blocks of about 1 mm3) werexed for 4 h by immersion in icecold 1.5% glutaraldehyde (Polysciences, Inc., War-ington, PA, USA) buffered with 0.07 M cacodylate buffer (pH 7.4), containing 7%ucrose, followed by post-fixation in OsO4 (Sigma Chemical Co., St. Louis, MO, USA)n 0.1 M cacodylate buffer (pH 7.4) for 2 h at 4 ◦C, dehydrated in a graded series ofthanol and embedded in Epon 812.

For light microscopy pre-examination, semithin sections (1 micrometre thick)ere stained with 1% borated methylene blue. For electron microscopy, ultrathin

ections (about 600 A thick) were cut from the blocks, mounted on uncoated 200-esh-copper grids, and doubly stained with saturated uranyl acetate in 50% acetone

nd Reynold’s lead citrate solution. The specimens were examined with a Zeiss EM00 electron microscope operating at 80 kV.

.4. Statistical analysis

Isoprostane data were presented as means ± standard deviation (SD) for nor-ally distributed variables. Differences between groups were evaluated using

ndependent-sample t test (continuous normally distributed data). Two-tailed p val-

es of less than 0.05 were considered significant. In all graphs, error bars representhe standard deviation of the mean.

Differential immunolabeling expression data were not normally distributed;herefore, the Kruskal–Wallis nonparametric test was used. Statistical significances indicated with a *(p value < 0.05).

(*).

3. Results

3.1. Lung and plasma F2-isoprostanes

In lungs, total F2-IsoPs levels were not modified at the earliertime points (24 h and 7 days) after either treatments (CdCl2 orSiNPs-Cd), while significant increased (56% and 43%) levels wereobserved after 30 days in both CdCl2 and SiNPs-Cd groups, respec-tively, compared to controls (32.8 ± 7.8 ng/g) (Fig. 1, mean ± SD):notably, F2-isoprostane enhancements were similar in these twogroups.

A parallel increase of free F2-IsoPs levels was observed inplasma samples (by 94.9% and 79.5%) of CdCl2 and SiNPs-Cd groups,respectively, compared to controls (29.1 ± 9.8 pg/ml) with effectsnoticeable at the earlier time point (7 days) and lasting until 30 days(increase of 112.7% and 95.1%, in CdCl2 and SiNPs-Cd group, respec-tively, compared to control group 28 ± 8 pg/ml) (Fig. 2). Again, theF2-IsoPs data were not significantly different between groups (i.e.CdCl2 and SiNPs-Cd) at both time points post exposure (i.e. 7 and30 days).

After treatment with SiNPs (600 �g/rat, i.t.), the lung and plasmaF2-IsoPs levels were similar to those observed in the control groupat all time points considered (Figs. 1 and 2).

Fig. 2. Free F2-Isoprostane levels in plasma. Plasma F2-IsoPs levels have been evalu-ated at 24 h, 7 and 30 days after i.t. treatment with CdCl2, SiNPs, and SiNPs-Cd. Dataare means ± standard deviation (SD). Differences between groups were evaluatedusing two-tailed t test and p value < 0.05 was considered statistically significant (*).

206 T. Coccini et al. / Toxicology 302 (2012) 203– 211

Fig. 3. Lung histology. Light microscopy (H&E) in control lungs (a) and some structural alterations detected in pulmonary parenchyma of SiNPs-Cd-treated rats after 7 daysf bronca d alved (a–c).

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rom i.t. instillation (b and c). (a) Normal alveolar and bronchiolar morphology; (b)rrowhead, respectively) in the peribronchiolar and perivascular area; (c) collapseistortion and epithelial cell desquamation (arrow); Objective magnification: x 40

.2. Lung histology

The lung parenchyma, analyzed by both light and electronicroscopy, after i.t. exposure to all the different treatments,

.e. CdCl2, SiNPs-Cd and SiNPs, clearly showed patterns of injury,ith diverse intensity degrees, characterized by collapsed alveoli,

hickening of alveolar wall and micro-hemorrhagic foci, fre-uently accompanied by bronchiolar deformation and epithelialell desquamation (Fig. 3, b and c). Noticeably, the development ofronchus-associated lymphoid nodule along with granulomatousormation in the peribronchiolar and perivascular regions were alsoetected (Fig. 3b), together with the presence of several inflamma-ory areas. These overall effects were observed at 7 days and lastedntil the 30th day.

These morphological alterations were paralleled by changes inhe expression of some antioxidant proteins, e.g. SOD1, iNOS andOX-2, as reported below.

.3. Immunocytochemistry

The localization and distribution of SOD1, iNOS and COX-2,ssentially involved in oxidative stress pathway, revealed an exten-ive spreading in bronchiolar and alveolar cells, as well as in theapillary component, evidencing the pulmonary reaction to thenjury, already observable at 7 days after i.t. exposure, and lasting

ntil 30 days (Table 1), with CdCl2 and SiNPs-Cd treatments causingdverse effects of comparable extent. SOD1-, iNOS- and COX--immunoreactivity significantly enhanced in a time-dependentanner (7 days <30 days), after both CdCl2and SiNPs-Cd (Fig. 4:

able 1xpression of immunolabelling for SOD1, iNOS and COX-2 on a semiquantitativevaluation in lungs of control, CdCl2-, SiNPs- and SiNPs-Cd-treated rats, 24 h, 7 and0 days after i.t. exposure.

Control 24 h 7 days 30 days p value

SiNPs-CdSOD1 ± ± ++ ++± *iNOS ± + ++ ++ *COX-2 ± ± ++ ++± *

CdCl2SOD-1 ± ± +± ++ *iNOS ± ± +± ++ *COX-2 ± ± ++ ++± *

SiNPsSOD1 ± ± + +± NSiNOS ± ± +± +± NSCOX-2 + ++ ++ +± NS

egree of staining intensity: from undetectable (−) to strong (++++). values calculated by Kruskal–Wallis test: (*) <0.05.

hus-associated lymphoid nodule along with granulomatous formation (arrow andoli with wall thickening and micro-hemorrhagic foci, associated with bronchiolar

b–c, f–g and l–m, respectively). SiNPs did not significantly influenceany of the investigated molecules (Table 1; Fig. 4d, h, n).

Noticeably, numerous SOD1-immunopositive activatedmacrophages were detected in the bronchiolar and alveolarepithelium of treated animals, particularly evident in collapsedareas, appearing heavily labelled (Fig. 4, b and c), being also observ-able at ultrastructural level, at which several eterophagosomes,containing internalized material in different stages of degradation,were clearly evident (Fig. 5, a and b).

The strongest iNOS-immunopositivity was detected after SiNPs-Cd as well as after CdCl2, at the capillary level, associated with thepresence of several immunopositive inflammatory cells (Fig. 4, fand g).

A marked COX-2-immunoreactivity was detected both at thecapillary and bronchiolar levels (airway epithelium) particularlyafter SiNPs-Cd and CdCl2 (Fig. 4, l and m), in correlation with thealveolar collapsed status.

The marked increase in SOD1, COX-2 and iNOS immunola-belling, with different expression patterns in bronchiolar, alveolarand vascular epithelium, observed after either CdCl2 or SiNPs-Cdtreatment (Table 1), is consistent with (i) the integral and pivotalrole played by both SOD1 and the two cytokine-inducible enzymesin pulmonary system, particularly in the development and progres-sion of lung injury, and (ii) the known function of the respiratoryepithelia as the first line of defence after insult, being also in accor-dance to previous in vitro and in vivo findings related to cadmiumtoxicity.

4. Discussion

Reactive oxygen species (ROS) producing lipid peroxidation andDNA damage have been implicated in cadmium toxicity (Liu et al.,2009; Morales et al., 2006). Recent investigations in rats exposed tocadmium indicated Cd-induced lipid peroxidation in several organs(Galazyn-Sidorczuk et al., 2011) and dose-dependent changes inseveral oxidative stress markers including F2-IsoPs (Rogalska et al.,2009). F2-IsoPs include a series of prostaglandin F2-like compoundswhich are generated by free radical-catalyzed peroxidation ofphospholipid-bound arachidonic acid, through a cyclooxygenase-independent pathway (Milatovic et al., 2011). Initially formedin situ on phospholipids (esterified F2-IsoPs), F2-IsoPs are releasedinto the circulation as free F2-IsoPs and, owing to their relativelylow reactivity, can be easily measured in biological samples (e.g.plasma) as oxidative stress markers (Milne et al., 2005).

In our study, oxidative stress was observed in lung tissuesand plasma after both CdCl2 and SiNPs-Cd treatments. The toxicresponse was reflected by elevated levels of F2-IsoPs and pul-monary over-expression of SOD1, COX-2 and iNOS extensively

T. Coccini et al. / Toxicology 302 (2012) 203– 211 207

Fig. 4. Immunostaining patterns of SOD1, iNOS, and COX-2 expression in rat pulmonary tissues. Immunocytochemical labelling for SOD1 (a–d), iNOS (e–h), and COX-2 (i–n)in lungs of control (a, e and i), CdCl2- (b, f and l), SiNPs-Cd- (c, g, m), and SiNPs-treated rats (d, h, n), 7 and 30 days after i.t. exposure. The SOD1 expression, mainly localizedat alveolar macrophages level, was clearly detectable after both CdCl2 and SiNPs-Cd treatments, still at 7 (b arrows) and lasting until 30 days (c arrows), respectively. Thei lveolaa fter ba n).

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NOS labelling was primarily observed in the inflammatory cells surrounding the arrows). The COX-2 expression was markedly increased at 7 as well as at 30 days, and epithelial levels of the alveolar wall (arrows). Objective magnification: x 40 (a-

pread out in bronchiolar and alveolar cells, as well as in the capil-ary component.

In SiNPs-Cd-treated animals, the pattern of change in iso-rostane levels was organ- and time-dependent: in lung, changesere not evident until 4 weeks after dosing; in plasma, F2-IsoPs

evels were already increased at day 7 and were still enhanced0 days after treatment. Similarly, elevated plasma F2-IsoPs levelsere found in rats 7 and 30 days after i.t. instillation of CdCl2.

The results indicated comparable oxidative stress response inung tissues after administration of cadmium as inorganic metal or

d-nanoparticles. Pulmonary changes were delayed in onset andere preceded by marked increase of F2-IsoPs levels in plasma

uggesting that peripheral plasma may be a sensitive target for

ig. 5. Lung ultrastructural morphology. (a and b) Electron microscopy of activated alvencidence of cytoplasmic phagosomes and residual bodies (arrows). Original magnificatio

r capillary wall, both at 7 and 30 days after CdCl2 and SiNPs-Cd instillation (f and goth CdCl2 and SiNPs-Cd (l and m) compared to controls, localized both at capillary

Cd-induced lipoperoxidation and that plasma F2-IsoPs may be earlypredictive indicators of later pulmonary oxidative insult. In pre-vious studies, elevated plasma levels of F2-IsoPs were found insubjects suffering from respiratory tract disorders as well as inexperimental in vivo models of oxidative stress (Comporti et al.,2008, 2009). Notably, elevated F2-IsoPs levels were also reportedin individuals exposed to environmental agents such as ozone,cigarette smoke, and allergens (Comporti et al., 2008; Janssen,2008). While measurement of F2-IsoPs is increasingly being usedas an indicator of lung disease state and severity (Comporti et al.,

2008; Janssen, 2008), very little is known about the time-course oflung tissue isoprostane accumulation and the correlation betweenchanges in isoprostane levels and the onset/progression/regression

olar macrophages (AM), 30 days after SiNPs-Cd treatment, characterized by a highn: x 4400.

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f specific disease symptoms (Lakshminrusimha et al., 2006;anssen, 2008).

Several reports have indicated that, in addition to their impor-ance as indicator of oxidative damage, F2-IsoPs can also exertntrinsic biological effects by interacting with tissue receptorsnvolved in constriction of pulmonary vessels and airways (Kangt al., 1993; Comporti et al., 2008).

The expression of SOD1, an enzyme involved in cellular protec-ion from oxidative stress, was similarly increased in lung tissueamples from both SiNPs-Cd and CdCl2 groups mainly detectedn activated macrophages observable at bronchiolar and alveolarpithelial levels. Changes, seen at day 7, were still observable 30ays after treatment. According to the data showing Cd tendencyo induce metallothionein, increase cellular glutathione levels, andctivate antioxidant transcription factor Nrf2 (Liu et al., 2009),OD1 over-expression may represent a possible adaptive defenceechanism activated by cells in response to Cd-induced prooxida-

ive insult.Similarly to SOD1, the COX-2 and iNOS inducible enzymes,

ere highly expressed at day 7 until day 30 in pulmonary tis-ues of both SiNPs-Cd and CdCl2 groups. Specifically, COX-2 wasound to be over-expressed in treated rats both in vascular andronchiolar epithelia accompanied with an evident alveolar col-

apsed status. This enzyme, a mitogen-inducible isoform criticallynvolved in pathogenesis of lung diseases (Park and Christman,006), usually expressed at minimal levels under normal condi-ions (Radi et al., 2010), is elevated during inflammation catalyzingrostaglandin synthesis (i.e. PGE2), and it is also induced by oxida-ive stress mediated by NF-kB (Nuclear Factor-KappaB) (Lee et al.,006). Particularly, COX-2 was shown to up-regulate different pul-onary cell types including endothelial and inflammatory cells

DuBois et al., 1998; Kundu et al., 2009). Regarding to iNOS, itas mainly detected at capillary level and in some inflammatory

ell clusters after both SiNPs-Cd and CdCl2 treatments. A high-utput induction of this enzyme has been shown to occur in anxidative environment, and high levels of nitric oxide (NO), pro-uced by iNOS as a defense mechanism, may react with superoxide

eading to peroxynitrite formation and cell toxicity. In particu-ar, in vitro and in vivo findings have shown a specific rise inOX-2 and iNOS expressions after exposure to Cd (Morales et al.,006; Park et al., 2008). Moreover, these two inducible enzymesCOX-2 and iNOS) have been reported to be over expressed in dif-erent models of oxidative stress in vivo (Swierkosz et al., 1995;ane et al., 1994). Many studies associate oxidative stress with

nflammation, a relationship that has also been described for Cd-nduced pulmonary inflammation (Kirschvink et al., 2006; Stosict al., 2010). Accordingly, our recent in vivo investigation indi-ated that not only CdCl2 but even SiNPs-Cd are able to induceung damage characterized by morphological alteration as well ashe occurrence of inflammation (accompanied by granuloma for-

ation) and stromal fibrogenetic reaction (Coccini et al., in press).pecifically, expression changes of several molecules, e.g. differ-nt cytokines/chemokines and metabolic factors, were observedcutely (24 h after i.t.) and lasted until the 30th day. SiNPs-Cd treat-ent produced a more marked effect compared to SiNPs and CdCl2.n increased pro-inflammatory cytokines/chemokines (i.e. IL-6,

P-10, and TGFß1) immunoreactivity revealed an intensive spread-ng in bronchiolar, alveolar (e.g. endothelium and macrophages)nd stromal cells. Parallely, diffuse collagen fibers deposition wasetected both in juxta-bronchiolar areas and within the alveolaralls (Coccini et al., in press).

Furthermore, we also demonstrated enhanced apoptotic

henomena followed by a consequent increased cell prolifera-ion after acute i.t. insult (Coccini et al., in press) as result of aignificant cellular turnover aimed at tissue repair/lung homeo-tasis (Alenzi, 2004; Li et al., 2004). This pulmonary insult was

302 (2012) 203– 211

induced by both CdCl2 and SiNPs-Cd treatments for which wepresently demonstrated an oxidative stress response. Accordingly,many reports have documented the inflammation-mediated oxida-tive damage following NP exposure also remarking that oxidativestress may act as a critical mechanism that links inflammation,excessive extracellular matrix deposition and lung cell apoptosis(Donaldson et al., 2005; Li et al., 2008; Park and Park, 2009; Petracheet al., 2008; Stone et al., 2007). Several experimental findings havesuggested that oxidative stress may play an important role inthe pathogenesis of pulmonary tissue fibrosis, affecting apoptosisand proliferation of both structural and inflammatory cells, alsoaltering the cytokine microenvironment balance. Indeed, oxidativestress-mediated activation of pro-inflammatory cytokines has beenshown to cause TGFß1 release, which may function as a masterswitch in tissue repair and wound healing, inducing lung fibro-blast proliferation thus leading to fibrogenesis (Mastruzzo et al.,2002; Willis et al., 2005; Agostini and Gurrieri, 2006). The complexrelationship between oxidative stress and inflammation is a highlydebated issue, being also a topic research area in particle toxicology(Donaldson et al., 2005; Li et al., 2008; Stone et al., 2007). Whetherthe inflammatory or the oxidative stress response occurs firstlyremain a critical question. Based on our experimental data, owingto the early detection of cytokines/chemokines increase (at 24 h),compared to the delayed overexpression of the three antioxidantproteins (i.e. SOD1, iNOS and COX-2) (at 7 days), the inflamma-tion response seemed to occur before the oxidative stress cascadeevents.

In relation to the Cd insult inflicted to the lung, extensive in vivomechanistic studies on pulmonary retention of inhaled (by i.t.)CdCl2, have shown that most of the total lung Cd (>90%) remainedin the lavaged lung, 10% in the lavageable cell fraction (mostlyalveolar macrophages), and about 4–8% in lung lavage fluid (super-natant) throughout a 30-day observation period (Oberdörster,1992; Oberdörster et al., 1979, 1994) in rats.

With respect to the SiNPs-Cd, the “nano” dimension may havefacilitated the cell concentration and lung toxicity of the adminis-tered cadmium. A “Trojan horse” type mechanism involving silicananoparticles as effective carriers for the cellular uptake of toxicmetals has been described (Park et al., 2010). In human lung epithe-lial cells, exposure to silica nanoparticles doped with metals suchas iron, manganese, cobalt or titanium was shown to generatehigher concentrations of reactive oxygen species and to inducemore severe oxidative stress compared to equivalent amounts ofthe respective metal ions (Limbach et al., 2007).

Though SiNPs are generally considered to be non-toxic, exper-iments using cell cultures or animal models have indicateddose-dependent cytotoxicity, increased reactive oxygen species,and reversible lung inflammation (Cho et al., 2007; Eom and Choi,2011; Kaewamatawong et al., 2006; Lee et al., 2011; Napierskaet al., 2010; Panas et al., 2012; Park and Park, 2009). Accord-ingly, our recent study demonstrated pulmonary inflammationand fibrosis after SiNPs instillation (Coccini et al., in press).Particularly, SiNPs induced overexpression of pro-inflammatorycytokines/chemokines, i.e. IL-6, IP-10, and TGFß1, as well as diffusecollagen deposition (e.g. fibrogenesis), already detected 24 h post-exposure, and persisted until the 30th day, with an extent similar tothat observed in CdCl2, but less markedly than in SiNPs-Cd. On theother hand, in the present study, differing from CdCl2 and SiNPs-Cd,SiNPs did not affect pulmonary SOD1, COX-2 and iNOS expressionsnor modify the physiological levels of tissue isoprostanes.

In summary, these results indicated delayed occurrence of pul-monary oxidative stress after i.t. instillation of both SiNPs-Cd and

CdCl2 in rats. The effects of CdCl2 and SiNPs-Cd on tissue iso-prostanes and pulmonary SOD1, COX-2 and iNOS were comparableand no changes involving these markers were observed in ani-mals treated with SiNPs. The data suggest a primary role of the

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admium moiety in the biological response to SiNPs-Cd althought seems unlikely that the changes produced by SiNPs-Cd adminis-ration merely reflected the action of cadmium ions released fromanoparticles. Indeed, chemical experiments with SiNPs-Cd haveemonstrated limited release of cadmium ions from the nanopar-icles dispersed in physiological solution, the maximum metalelease being ca. 15% over a 10-day period. In addition, the ten-ency to form aggregates and agglomerates of these doped NPsCoccini et al., in press) may also have contributed in triggeringhe described SiNPs-Cd lung effects. In fact, agglomeration haseen shown to influence NP toxicity by affecting cellular uptakend biodistribution in vivo, modifying the specific surface areaLimbach et al., 2007; Gosens et al., 2010; Kim et al., 2010; Parkt al., 2010; Zook et al., 2011). Nanoparticle agglomeration is aasic process that results in a reduction of surface free energy by

ncreasing their size and decreasing their surface area. Individualanoparticles when agglomerated may create particles that are no

onger in the nanoscale size range. Whether those agglomeratedarticles retain toxic properties of the individual nanoparticles orre capable of subsequently de-agglomerating is a critical ques-ion (Balbus et al., 2007). While several studies have shown thatingle particles have the ability to form agglomerates in a bio-ogical matrix, it is not known whether agglomerated particlesould become single particles again when introduced in a bio-ogical system. Moreover, agglomeration state, as measured in airr water, may change inside the body through interactions withiological fluids and proteins and change further still within differ-nt tissue/cellular/subcellular compartments that define differentptimal affinities for the particles and biological macromolecules.nterfacial effects of NPs are dependent on both the original NPurface and surrounding system (leading to a “NP coronas” formedround the NPs in biological systems and subsequent effects oniological behavioural) (Kendall and Holgate, 2012). In the presenttudy, the DLS data demonstrated an agglomeration and aggrega-ion extent of SiNPs-Cd (about 350 nm) greater than that measuredor SiNPs (about 120 nm) in physiological solution. For instance,he presence and agglomeration state of these two types of NPsere not investigated at pulmonary tissue or subcellular levels in

at. On the other hand, SiNPs-Cd affected both inflammatory andxidative stress pathways differently from SiNPs causing inflam-atory responses only (Coccini et al., in press). Altogether, these

esults show a higher SiNPs-Cd reactivity (regardless of whetherorm type is present: original, agglomerate, or with sorbed materialt NP surface) than SiNPs in the lung tissue.

Furthermore, observations from toxicogenomic studies in ratsCoccini et al., 2011, 2012) indicated remarkable differences in theene expression profiles observed in lung and secondary organsfter administration of SiNPs-Cd or comparable amounts of CdCl2.ur previous transcriptomics study indicated that in lung only fewenes were regulated at 7 day after CdCl2 and SiNPs-Cd (22 and1 identified probes, respectively) compared to those modulatedt 30 days (138 and 156, respectively) (Coccini et al., 2011). Most ofhe genes modulated by SiNPs-Cd were different than those mod-lated by CdCl2 at 30 days. Applying figures-of-merit analysis, aluster containing genes upregulated only by CdCl2 was identi-ed: the categories enriched in this cluster were inflammation,lood coagulation, metabolic pathway and phagocytosis (includ-

ng genes of cytochrome P450 and coagulation factors). A clusterhowing genes upregulated only by SiNPs-Cd was also identified:his group was enriched in genes involved in the inflammatoryrocess which includes several chemokines. The overall genomicata show a complex regulation induced by SiNPs-Cd, only partially

verlapping with those of CdCl2, but modulating some relevantpecific pathways. Combining the gene expression results (Coccinit al., 2011) with biochemical and immunohistochemical evalu-tion at the pulmonary tissue (Coccini et al., in press) could be

302 (2012) 203– 211 209

profitable in providing more direct linkages between a change ingene expression and the type and outcome of the toxic response;however, this link does not exactly emerge when comparing ourdata. In fact, regarding the inflammation and metabolic pathways,our immunohistochemical results (Coccini et al., in press), showedthe occurrence of increased pulmonary cytokines- (i.e. IL-6, TGFß1,and IP-10) and CYP450-immunoreactivity, after both CdCl2 andSiNPs-Cd. On the other hand, clusters populated by genes that weredown- or up-regulated by both stimuli (CdCl2 and SiNPs-Cd) werealso identified by genomic analysis: they included genes involvedin cell cycle regulation and genes relevant in unfolding proteinresponse (UPR) (Coccini et al., 2011). The UPR is an essential adap-tive intracellular signaling pathway that responds to metabolic,oxidative stress, and inflammatory response pathways (Wang andKaufman, 2012). In conditions of prolonged stress, the goal of theUPR changes is from being one that promotes cellular survival toone that commits the cell to a pathway of apoptosis. Accordingly,in our study, lung damage evaluation in terms of apoptosis (byTUNEL staining) and cell proliferation (by PCNA immunoreactiv-ity), indicated enhanced apoptotic phenomena by both SiNPs-Cdand CdCl2, followed by a significant increase of proliferating cells(Coccini et al., in press). In addition, the present study supportsthe capacity of both CdCl2, and SiNPs-Cd to cause also long-lastingoxidative stress following pulmonary exposure in rat.

Conflict of interest statement

The authors declare they have no proprietary, financial or per-sonal interest of any kind or nature that could be construed as beinga conflict of interest.

Acknowledgements

Work was supported by Grants from European Commission,Italian Ministries of Health, Research and Education, and CARIPLOFoundation (Rif. 2011–2096). The authors wish to acknowledge Mr.Davide Acerbi for his excellent technical assistance.

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