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American Journal of Public Health Research
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The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

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The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

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Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
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The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

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An integrated in Vitro and in Vivo Testing Approach to Assess Pulmonary Toxicity of Engineered Cadmium-Doped Silica Nanoparticles

Uliana De Simone, Elisa Roda, Cinzia Signorini, Teresa Coccini*

Laboratory of Clinical and Experimental Toxicology, Toxicology Division, IRCCS Salvatore Maugeri Foundation, Scientific Institute of Pavia Medical Centre, Pavia, Italy

Abstract: An in vitro and in vivo testing strategy for assessing the pulmonary effects was used to investigate the safety characteristics of silica nanoparticles doped with cadmium (Cd-SiNPs). In A549 cells, Cd-SiNPs (0.5-100 μg/ml) caused (i) mitochondrial dysfunction and apoptosis at 1 μg/ml, (ii) GSH depletion at 10μg/ml, (iii) membrane alterations at 25 μg/ml, after 1-day, and (iv) cell growth and proliferation inhibition at 0.05 μg/ml after prolonged exposure. Cd-SiNP effects were more pronounced compared to CdCl₂. SiNPs affected GSH content only. In vivo results revealed early (1 day) and persistent (until 1 month) rat lung damage after intratracheal instillation of Cd-SiNPs (1mg/rat) in terms of enhanced apoptotic phenomena and altered lung parenchyma morphology. Cd-SiNPs and CdCl₂ caused a delayed occurrence of oxidative stress by increasing SOD1, iNOS, and F2-IsoPs. The latter was preceded by marked increase of F2-IsoPs levels in plasma. SiNPs did not cause oxidative stress. Cd-SiNPs showed a higher reactivity than CdCl₂ and SiNPs. In vitro and in vivo data on Cd-SiNP toxicity suggest that the lung is a susceptible target tissue. These findings support the concept that multiple assays and an integrated testing strategy should be recommended to characterize toxicological response to NPs.

Keywords: pulmonary, in vivo, in vitro, nanotoxicology, silica, cadmium

Citation: Uliana De Simone, Elisa Roda, Cinzia Signorini, Teresa Coccini, An integrated in Vitro and in Vivo Testing Approach to Assess Pulmonary Toxicity of Engineered Cadmium-Doped Silica Nanoparticles. American Journal of Nanomaterials, 3 (2). 40-56. January 2015. doi:10.12691/ajn-3-2-1

1. Introduction

Today there are considerable uncertainties on how to assess exposure to nanoparticles (NPs), and conventional procedures for assessing chemical toxicity seems not completely suitable for application to NPs [1]. As there is not yet a generally applicable paradigm for nanomaterial hazard identification, a case-by-case approach for the risk assessment of nanomaterials is still warranted [2]. Several Federal agencies [3,4,5] and regulated communities [6] have initiated focused efforts to study the potential risks of exposure to nanoscale materials, and others have been continuing to conduct research that is expected to facilitate risk evaluation. The new research is intended to identify the relationship between the physicochemical characteristics and toxicological properties of nanoscale materials. The researchers at NIST (National Institute of Standard and technology) developed a multi-tiered testing approach to study the NP toxicology [7]. Toxicity testing should be initiated with a careful i) physic-chemical characterization (e.g., particle size, particle size distribution, reactivity, surface area, particle mass, impurity, and aggregation tendency) assisted by using reference materials that are increasingly available, then ii) well characterized NPs can be utilized in a tiered fashion by first using cellular (in vitro) models. In vitro studies allow to define and characterized toxicity pathways of NPs through higher throughput, replicates, and parallel tests. In addition, in vitro approach has the ability to test across large concentration ranges to develop extensive dose response relationships for various toxicity pathways; iii) subsequent step consists in a focused limited series of in vivo studies guided by information generated from in vitro investigations, designing appropriate exposure concentrations and defining the critical health endpoints to be monitored.

Despite of their unique advantages and applications in domestic and industrial sectors, use of materials with dimensions in nanometers has raised the issue of safety for workers, consumers, and human environment, and in this context inhalation is an important route of nanoparticle exposure. The lungs would be the first line of contact for particles that gained entry into the body and hence the most likely organ for accumulation and long-term exposure. Interest in the respiratory system as a target for the potential effects, both beneficial and adverse, of nanoparticles is reflected by the steady increase in the number of scientific publications on these subjects during the past decade [8]. Upon inhalation, NPs can penetrate deeper into lungs and interact with epithelium. This can cause inflammation and chronic effects by further penetration into interstitum and finally these NPs may transfer to lymph nodes [9,10]. Particle-cell and particle-tissue interaction increases with deeper penetration and this interaction promotes adverse health effects. Still, insoluble particles reside inside the lungs for prolonged time period, which may cause cells destruction and biological disorder of tissues [11,12]. Several in vivo [13,14] and in vitro [15,16,17] studies have indicated a range of toxic effects induced by NPs including increased reactive oxygen species and lung inflammation [18] as well as chronic pulmonary disease [19 for review]. Currently, there are no epidemiologic data indicating health hazards for the majority of nanomaterials.

Among the several types of engineered nanoparticles silica-doped metal nanoparticles have become increasingly important in the last decade, for example cadmium-doped silica nanoparticles have been developed for different applications including: informatics devices, drug delivery/activation, and fluorescence probes - quantum dots - for bioimaging [20,21,22]. Information on toxicity, exposure and health impact of these nanomaterials is still limited [20,23]. Some studies in animals have demonstrated mainly reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis after respiratory exposure to these NPs [24]. On the other hand a large body of evidence supports lung toxicity effects after cadmium exposure when inhaled [25,26]. The toxicity of Cd on human, has been investigated for over 50 years, and despite there are long terms studies the knowledge in this area is still expanding. Cadmium is a heavy metal of considerable environmental and occupational concern. Compounds are classified as human carcinogens by regulatory agencies [26] and the National Toxicology Program [27] based on epidemiologic studies showing a causal association with lung cancer and on inhalation studies in rodents. Currently, the risk associated with this type of nanoparticles (Cd-SiNPs) must be still carefully evaluated.

This paper analyzes the overall in vitro and in vivo toxicity results obtained by Cd-SiNP exposure. The assessment sustains, and thereafter have demonstrated, a valuable tiered model approach for NP toxicity evaluation using a lab-made NP model namely Cd-SiNP, as representative of materials that individually (i.e. silica NP and cadmium) have an extensive toxicity database. The toxicological testing design application shows an efficient toxicity testing by transitioning from quantitative and mechanistic toxicity testing in human cell line to qualitative, descriptive animal testing. Approach that allows the bulk of the screening analysis and high - volume data generation carried out in vitro, after which it follows limited, but critical, validation studies in animals.

The study considers the potential lung damage caused by exposure to silica nanoparticle (SiNPs) doped with cadmium (Cd-SiNPs) by an integrated approach applying a two-level strategy relied on in vitro and in vivo studies combination. In details, the integrated and complementary approach consisted in utilizing: i) A549 cell line as a standardized model to study human lung epithelium in vitro since these cells have many important biological properties of alveolar epithelial type II cells, such as membrane-bound inclusions, which resemble lamellar bodies of type II cells [28]. This cell line is routinely used as an in vitro model of pulmonary cuboidal epithelium to study the interaction of ambient air pollutants [29] including the ultrafine particles [30]; ii) rat animal model (Sprague-Dawley), as a species extremely suitable for the investigation of pulmonary toxicity and biokinetics parameter evaluations induced by exposure to environmental particles (e.g. fine, ultrafine and nano form).

In the first phase, a battery of in vitro high throughput cell-based assays was used to test well-characterized Cd-SiNPs. The testing strategy starts with the understanding the molecular mechanisms of toxicant effects of Cd-SiNPs on target cells (human A549 pneumocytes) by evaluation different endpoints, in terms, of apoptosis (by activated caspase-3 evaluation), metabolic activity (by MTT assay), membrane integrity (by calcein-AM/Propidium Iodide staining), oxidative stress (by GSH content evaluation), and growth and cell proliferation (by clonogenic assay) after exposure for 1 day or 10 days. In the successive tier, pulmonary toxic effects were assessed in focused in vivo experiment administering by intratracheal (i.t.) instillation of a Cd-SiNP single dose and evaluating the following endpoints after 1 and 7 days in the lung tissue: (i) apoptotic features (by TUNEL and electronic microscopy (EM)); (ii) histopathological analysis (by haematoxylin/eosin staining - H&E and EM), (iii), oxidative stress markers evaluating F2-Isoprostanes (F2-IsoPs) (by GC/NICI-MS/MS), SOD1 and iNOS (by immunochemistry). F2-IsoPs levels were also determined in plasma samples as a marker of oxidative stress in accessible matrix.

The toxicity of Cd-SiNP was compared to the effects produced by the SiNPs counterpart (not doped) or by CdCl₂.

The results have been used to make an overall evaluation of the in vitro findings in comparison with the toxic responses documented in vivo.

2. Materials and Methods

2.1. Chemicals

Silica nanosize (SiNP) was purchased from Degussa Gmbh (Germany) as HiSilTM T700, average pore size 20 nm, surface area 240 m²/g, pore specific volume of 0.4cm³/g. All reagents and chemicals for cell cultures, cadmium chloride hemi (pentahydrate; CdCl₂), and chemicals for Light and Electron Microscopy analyses were purchased from Sigma-Aldrich (Milan, Italy), except for Eukitt (Kindler, Freiburg, Germany) and glutaraldehyde (Polysciences, Inc. Warrington, PA, USA). The primary rabbit polyclonal antibody against Caspase 3 and Alexa 488-labeled antibodies were obtained from Molecular Probes (Life Technologies, Monza, Italy).

Primary rabbit polyclonal antibodies against SOD1 or iNOS were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An ABC kit containing a biotinylated anti-rabbit secondary antibody and an avidin biotinylated horseradish peroxidase complex were purchased from Vector Laboratories (Burlingame, CA, USA).

GSH quantification Kit was purchased from Oxis Interntional Inc. (Foster City, Ca, USA) and TUNEL assay from Oncogene Res. Prod. (Boston, MA, USA).

2.2. Synthesis and Physico-chemical Characterization of Engineered Cadmium-Doped Nanoparticles (Cd-SiNPs)

Synthesis and physico-chemical characterization of Cd-SiNPs were previously described in Coccini et al. [31]. Concisely, SiNPs were impregnated with cadmium nitrate dehydrate (CdNO₃ 3.56 × 10⁻² M) in an aqueous solution in which the silica was dispersed in a concentration ratio leading to a sample containing 40% Cd by weight. Then powder was subjected to grinding mills with high energy (200 rpm for 1.5 h, 400 rpm for 1.5 h, 600 rpm for 2 h) to get the most equal distribution of particle size and/or aggregates of particles. Elemental composition, shape, size distribution, and morphology of Cd-SiNPs were determined by dynamic light scattering (DLS), scanning transmission electron microscopy (STEM) technique and X-ray powder (XRD) diffraction. The release of cadmium from nanoparticles dispersed in physiological solution was determined as well as metal impurities by flame-atomic absorption analysis.

2.3. In Vitro Studies

2.3.1. A549 Pulmonary Cell Line

A549 clonal cell line from a human Caucasian lung adenocarcinoma with alveolar type II phenotype was obtained from ECACC (Sigma-Aldrich, Milan, Italy). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg streptomycin. Cells were maintained at 37 °C in in a humidified atmosphere containing 95% air and 5% CO₂.

Fresh stock solutions were prepared by dissolving test materials (SiNPs, Cd-SiNPs, and CdCl₂) in culture medium (DMEM) before each experiment, then cells were exposed to concentrations ranging from 0.05 to 100 μg/ml for 24 h or 10 days.

2.3.2. Short-term Exposure (1 day)

2.3.3. Metabolic Mitochondrial Function (MTT assay)

The MTT assay was used to evaluate mitochondrial activity. Cells were seeded in 96-well plates at density of 1 x 10⁴ cells/well in 100 μl complete medium/well the day before treatment, then the cells were exposed to Cd-SiNP, SiNP and CdCl₂ at final concentrations ranging from 1 to 100 μg/ml for 24 h at 37 °C. After incubation, the cell culture media were aspirated, 10 μl MTT (5 mg/ml) was added to each well and incubated for 3 h. Afterwards, the resulting formazan crystals were solubilized in 100 μl/well of DMSO and quantified by measuring absorbance at 550 nm by Biorad microplate reader.

Data were expressed as a percentage of control (untreated cells).

2.3.4. Membrane Integrity (Calcein AM/PI staining)

The membrane integrity and cell morphology were evaluated by the co-incubation of the double staining: 2 μM calcein-AM and 2.5 μg/ml Propidium Iodide (PI) for 10 min at 37 °C. Cells were examined under a Zeiss Axiovert 25 fluorescence microscope provided with a triple filter set (excitation: 400, 495, 570 nm; beamsplitter: 410, 505, 585 nm; emission: 460, 530, 610 nm), and combined with digital camera (Canon powershot G8). Viability was expressed as percentage cells retained calcein (green fluorescence) compared to the total cells counted (calcein-positive plus PI-positive (red fluorescence)).

2.3.5. Apoptotic Pathway: Immunofluorescence Detection of Activated Caspase 3

The protocol of caspase-3 activation was previously described in De Simone et al. [32]. Cells (2 x 10⁵) were exposed to increasing concentrations (1 - 50 μg/ml) of the tested materials for 24 h at 37 °C.

Then, the cells were fixed, rehydrated and incubated with polyclonal antibodies recognizing caspase-3 (dilution 1:200 in PBS). The bound antibodies were revealed with Alexa 488-labeled (dilution 1:100 in PBS). DNA was counterstained with 1 μg/ml PI, washed with PBS and mounted with Fluoroshield. Cells were examined under a CX41 Olympus fluorescence microscope (excitation conditions: 470 nm excitation (T% = 40), 505 nm dichroic beamsplitter, 510 nm long pass filter) combined with a digital camera (Infinity2). The images were captured using 100X objective lens and store on a PC.

2.3.6. Oxidative Stress: Quantitative Evaluation of Intracellular Glutathione (GSH)

Cells were seeded in six-well plates (5 x 10⁵ cells/well) and cultured overnight at 37 °C, then were exposed to increasing concentration (1 - 100 μg/ml) of Cd-SiNPs, CdCl₂ and SiNPs, for 24-h exposure. The cell pellet was resuspended in metaphosphoric acid, homogenized (Ultra Turrax, Janke & Kunkel), centrifuged (3000 g, 4 °C for 10 min), and then mixed with 4-chloro-1-methyl-7-trifluromethylquinolinium methylsulfate and 30% sodium hydroxide reagents. The absorbance was measured spectrophotometrically (Spectrometer Lambda Bio 20, Perkin Elmer) at 400 nm, total GSH content was determined with a standard curve.

2.3.7. Long-term Exposure (up to 10 days): Cell Growth and Proliferation

For the clonogenic assay procedure (detailed described in De Simone [33]) cells were seeded in six-well plates at density of 400 cells/well, each well containing 2 ml of cell culture medium. After attachment the cells were washed with 2 ml PBS, and treated with 2 ml of the tested materials (final concentration between 0.05 to 50 μg/ml) for 10 days (time period needed for the cells to form colonies). At the end of the treatment time the medium was aspirated, then the colonies were fixed, stained with Hematoxylin and counted for the evaluation of cell survival after the treatments. The colonies were examined under Zeiss Axiovert 25 microscope combined with a digital camera (Canon powershot G8). Digital photographs were stored on PC from each well using 2.5X objective lens. The number of colonies that arose after treatment was expressed in terms of plating efficiency (PE). PE was calculated by dividing the number of colonies formed by the number of cells plated per 100.

2.4. In Vivo Studies

2.4.1. Animals and Treatment by Intratracheal (i.t.) Instillation

All experimental procedures were performed in compliance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. All animals used in this research have been treated humanely according to the institutional guidelines, with due consideration for the alleviation of distress and discomfort.

Young, adult, healthy, male Sprague-Dawley rats (Charles River Italia, Calco, Italy; body weight 278±3 g) were housed in a humidity- (50±10% relative humidity) and temperature- (22 °C) controlled room. Rats were housed in individually ventilated plastic cages with hardwood chip bedding, fed ad libitum with a standard diet (4RF21), and provided with filtered water. They were maintained on a 12-h light:12-h dark cycle (lights on at 07.00 h). Rats were allowed to acclimate to the facility for a minimum of 2 weeks prior of experimental procedure.

For the treatment, groups of rats (n=6 total for each treatment group at each time point) were anesthetized with pentobarbital sodium for veterinary use and treated with a single i.t. (100 μl) of: i) Cd-SiNP (1 mg/rat, corresponding to about 250 μg Cd/rat), (ii) SiNPs (600 μg/rat), iii) CdCl₂ (400 μg/rat). The control group was instilled with 100 μl saline solution. In previous studies [34,35], a single i.t. instilled dose of 400 μg CdCl₂/rat was shown to induce in rats moderate lung injury evolving into chronic inflammation and fibrosis.

Just before i.t. exposure, the different NP suspensions were prepared by vortexing the suspension on ice to further force NP dispersion, avoiding the tendency to agglomerate and the formation of aggregates, without using any surfactants or solvents. The suspensions of the test materials were immediately used for the treatment.

The rats were sacrificed on 24 h, 7 and 30 days after treatment. Treated and control animals were deeply anesthetized with an overdose i.p. injection of 35% chloral hydrate (100 μl/100 gb.w.), and divided into two different sets. From a set of rats (n=3 for each treatment group), lungs were removed and blood collected for the isoprostane analyses. The blood samples in heparinized tubes were centrifuged at 2400 g for 15 min at 4 °C; the platelet-poor plasma was saved and the buffy coat was removed by aspiration.

From the remaining set of animals (n=3 for each treatment group), histological evaluation (by light and electron microscopy analyses), TUNEL assay, and immunohistochemically examining of SOD1 and iNOS in lung tissue were performed after vascular perfusion of fixative. Briefly, laparotomy was carried out, allowing to cannulate the pulmonary artery via the ventricle, and to insert an outflow cannula into the left atrium. Quickly, the tracheal cannula was connected to about 7 cm H₂O pressure source to inflate the lungs with air, and a clearing solution (saline with 100 U/ml heparin, 350 mosM sucrose) was perfused via the pulmonary artery, finally followed by the fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). After fixation, the lungs were carefully removed.

2.4.2. Lung Tissues

The top and the bottom regions of the right lungs of control and differently treated animals were dissected. Tissue samples were obtained according to a stratified random sampling scheme which is recommended to reduce variability of the sampling means, compensating the regional differences existing in the lung [36]. Next, the tissues were washed in NaCl 0.9% and post-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 7 h, then dehydrated through a graded series of ethanol and finally embedded in Paraplast.

Eight μm thick sections of the samples were cut in the transversal plane and collected on silan-doped slides for both histopathological evaluation (H&E) as well as immunocytochemical analysis.

2.4.3. Apoptotic Phenomena: TUNEL Assay

Apoptotic cell death was assayed by in situ detection of DNA fragmentation using the terminal deoxynucleotidyltransferase (TUNEL) assay. The lung sections were incubated with 20 μg/ml proteinase-K solution for 5 min, followed by 3% H₂O₂; after incubation with TdT/biotinylated dNTP and HRP-conjugate streptavidin (90 and 30 min at 37 °C, respectively), the reaction was developed employing 0.05% 3-amino-9-ethylcarbazole (AEC) in 0.1 M TRIS buffer (pH 7.6) with 0.2% H₂O₂, or a 0.1% DAB solution. Light nuclear counterstaining was achieved using Haematoxylin. The TdT incubation was omitted in some sections as negative control; in these conditions no staining was observed. The evaluation of TUNEL-cytochemically positive cells (TUNEL L.I.) was calculated as the percentage (Labelling Index) of a total number (about 500) of cells (bronchiolar, alveolar and stromal of the lung parenchyma), for each experimental condition, in a minimum of 10 randomly selected highpower microscopic fields. The slides were observed and scored with a bright-field Zeiss Axioscop Plus microscope. The images were recorded with an Olympus Camedia C-2000 Z digital camera and stored on a PC running Olympus software.

2.4.4. Electron Microscopy

Small lung blocks (about 1 mm³) were fixed in icecold 1.5% glutaraldehyde buffered with 0.07 M cacodylate buffer (pH 7.4), containing 7% sucrose, followed by post-fixation in OsO₄ in 0.1 M cacodylate buffer (pH 7.4), dehydrated in a graded series of ethanol and embedded in Epon 812. Ultrathin sections (about 600Å thick) were cut from the blocks, mounted on uncoated 200-mesh-copper grids, and doubly stained with saturated uranyl acetate in 50% acetone and Reynold’s lead citrate solution. The specimens were examined with a Zeiss EM 300 electron microscope operating at 80 kV.

2.4.5. Histopathology

Pulmonary tissues were also processed for histopathological analysis; specifically, lung sections of control and treated rats were stained with H&E to evaluate overall tissue structural changes.

2.4.6. Oxidative Stress Evaluation

2.4.6.1. SOD1 and iNOS in Lung: Immunohistochemical Investigation

To circumvent staining differences due to slight procedure changes, the reactions were carried out simultaneously on slides of control and treated animals at all stages. Immunocytochemistry was performed using commercial antibodies against SOD1 and iNOS isozymes, to demonstrate and localize the presence/distribution of markers of inflammation-related oxidative stress. The sections were incubated overnight at room temperature with the primary rabbit polyclonal antibody against SOD1 or iNOS, diluted 1:100 in PBS. Biotinylated anti-rabbit secondary antibody and an avidin biotinylated horseradish peroxidase complex were used to reveal the sites of antigen/antibody interaction. The 3,3’-diaminobenzidine tetrahydrochloride peroxidase substrate was used as chromogen, and Haematoxylin was employed for nuclear counterstaining. Then, the sections were dehydrated in ethanol, cleared in xylene, and finally mounted in Eukitt. As negative controls, some sections were incubated with phosphate-buffer saline in absence of the primary antibody. No immunoreactivity was observed in this condition.

Grading of labeling: A scoring system was used to evaluate the degree of immunostaining, using conventional bright-field microscopy according to a semiquantitative scale ranging from undetectable (−) to strong (++++). The localization and intensity of labeling was recorded and graded, with approximate percentages indicating those numbers of relevant cells showing intense positive reaction, as follows: (−) absent/undetectable immunohistochemical reaction; (+) mild positivity involving 1-10% of organ cells; (++) moderate immunoreactivity involving up to approximately 25% cells; (+++) strong immunopositivity involving up to approximately 50% cells; (++++) maximal immunohistochemical reaction involving more than approximately 50%. The slides were observed and scored with a bright-field Zeiss Axioscop Plus microscope. The images were recorded with an Olympus Camedia C-2000 Z digital camera and stored on a PC running Olympus software.

2.4.6.2. F2-IsoPs in Plasma and Lung

Free and total (sum of free plus esterified) F2-IsoPs were determined by gas chromatography/tandem mass spectrometry (GC/NICI-MS/MS) in plasma and lung samples, respectively, as described by Signorini et al. [37,38]. In previous studies [37,38,39,40,41], GC/NICI-MS/MS has proved to be a reliable procedure (in term of specificity, repeatability and accuracy) to assess F2-IsoPs as indicators of free radical-induced lipid peroxidation.

2.5. Statistical Analysis

In vitro study: Data from acute exposure were obtained from three independent experiments each carried out in six replicates. Data from prolonged exposure were obtained from two independent experiments and each experiment was carried out in three replicates. Results are expressed as mean ± standard deviation (SD). Statistical significance was assessed by one-way ANOVA followed by Tukey’s test. A value of P < 0.05 was considered statistically significant.

In vivo study:

- For histology and immunocytochemistry: TUNEL data were analyzed by two-way analysis of variance (ANOVA) followed by the bonferroni test for multiple mean comparison. Differential immunolabeling expression data are not normally distributed so the Kruskal-Wallis nonparametric test was used. Statistical significance is indicated with P value < 0.05.

- Isoprostane data are presented as means ± SD for normally distributed variables. Differences between groups were evaluated using independent-sample t test (continuous normally distributed data). Two-tailed P values of less than 0.05 were considered significant. In all graphs, error bars represent the SD of the mean.

3. Results

Comparing the in vitro and in vivo results may help to assess the concordance/discordance between the alternative methods and the in vivo methods, and to test the predictability of the alternative methods for the in vivo results [42]. Table 1 summarizes the global results.

3.1. Characteristics of Cd-SiNPs

Table 2 reports the major characteristics of the Cd-SiNPs. Quantitative STEM showed the morphology of Cd-SiNPs and the analysis of the elements by HAADF mode and EDS spectra confirmed the presence of Cd, Si and O. Cadmium and silica contents in the nanoparticles were 32.5% and 24.1% respectively, the particles presented spherical form, primary particle size range of 20-80 nm and specific surface area of about 200 m²/g. DLS showed tendency to form aggregates and agglomerates of about 350 nm (zeta potential of - 23 mV, in deionized water or DMEM). The release of cadmium from nanoparticles dispersed in physiological solution and in culture medium was 15% and 28%, respectively, after 16 h, and it was negligible in the subsequent 10 day period for both preparations.

Metal impurities were: Ca 0.3%, Na 0.2%, K 0.2%, Fe 0.04%, and Mn 0.001%, other metals werepresent in quantities less than 1%.

3.2. In vitro Results

Cytotoxicity (mitochondrial function, membrane integrity and cell morphology), apoptosis, oxidative stress and the capacity to form colonies of A549 cells are reported and compared after 1- and 10-day exposure to increasing concentrations (from 0.05 to 100 μg/ml) of SiNPs, Cd-SiNPs and CdCl₂.

3.2.1. Mitochondrial and Cell Membrane Cytotoxicity Evaluation

The A549 cytotoxicity was assayed in parallel by two colorimetric methodologies: MTT assay enables accurate, straightforward quantification of changes in metabolic activities (i.e. mitochondrial function) by spectrophotometric determination, and calcein-AM/PI staining allows qualitative evaluation on membrane integrity and cell morphology and quantitative evaluation on cell viability (cell live/cell death) by fluorescence microscopy.

Table 1. Summary of the in vitro and in vivo results with well characterized NPs (Cd-SiNP, SiNP) and CdCl₂

Table 2. Physico-chemical characteristics of the nanoparticles

MTT data: SiNP treatments did not show any significant cytotoxic effect for all tested doses (1 - 100 μg/ml) (Figure 1). On the contrary, the cells treated with Cd-SiNP and CdCl₂ after 24 h showed the mitochondrial function strongly affected, in particular Cd-SiNPs induced loss of cell viability (about 40%) already at the lowest dose tested of 1 μg/ml (Figure 1), while the cytotoxic effect of CdCl₂ treatment was detected at 25 μg/ml with about 20% mortality (Figure 1). A strong decreased viability was reached at doses ranging from 50 to 100 μg/ml: about 65-70% and 45-60%, for both Cd-SiNP and CdCl₂ treatments, respectively (Figure 1).

Figure 1. Mitochondrial metabolism by MTT in A549 cells after 1-day treatment. Mitochondrial metabolism by MTT assay in A549 cells exposed to increasing concentration (1 - 100 μg/ml) of Cd-SiNPs, CdCl₂ and SiNPs after 24 h. Data are mean ± SD of three separate experiments each carried out in six replicates. * Different from control 𝑃 < 0.05, Statistical analysis by ANOVA. Error bars: ± SD

Calcein-AM/PI data: SiNP treatment didn’t cause cell mortality in that the green fluorescence was uniformly diffused into cytoplasm for any concentrations tested (1 - 100 μg/ml). While, Cd-SiNP and CdCl₂ treatments induced dose-dependent cytotoxic effects (Figure 2). Cell viability loss by Cd-SiNP started 25 μg/ml, as evidenced by the presence of numerous red coloured cells (indicating damage to the cell membrane) which became markedly evident at the highest concentrations of 50 and 100 μg/ml with pronounced morphological cell alteration (Figure 2a, b): at doses ranging from 25 to 100 μg/ml, the decrease of cell viability was about 25-95%.

Figure 2. Cell membrane cytotoxicity evaluation by Calcein-AM/PI staining in A549 cells after 1-day treatment. (a) Representative images of randomly selected microscopic fields of A549 cells stained with calcein-AM/PI after 24 h exposure to increasing concentration (1 - 100 μg/ml) of Cd-SiNPs, CdCl₂ and SiNPs. There is a strong decrease of viability (low green fluorescence, and red fluorescence indicating cell death) dose-dependent cytotoxic effect in both Cd-SiNPs and CdCl₂ treatment groups. While, green fluorescence is uniformly diffused in treated SiNP cell at the all doses tested. Scale bar: 100 μm. (b) Semi-quantitative analysis of selected microscopic fields of A549 cells after 24 h exposure to increasing concentrations of Cd SiNPs, CdCl₂ and SiNPs (1 - 100 μg/ml), in terms of cell counts and expressed as percentage of live cells (mean ± SD, * Different from control 𝑃 < 0.05, Statistical analysis by ANOVA)

CdCl₂, caused cell viability loss starting at 50 μg/ml (about 50% decrease) (Figure 2a, b). In accordance with data from MTT reduction, the results of our studies confirm that PI positive (red) staining was increased in A549 cells exposed to both CdCl₂ and Cd-SiNPs evidencing cells damaged / dead in 24 h-post treatment period. PI is a fluorescent vital dye that stains DNA. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because they maintain plasma membrane integrity. In contrast those cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI. To assess the extent and mode of early or late apoptotic events induced by our test materials, caspase-3 activation protocol was applied.

3.2.2. Apoptotic pathway: Immunofluorescence Analysis of Activated Caspase-3

The apoptotic pathway was evaluated by antibodies against caspase-3 after 24 h exposure to increasing concentrations of SiNPs, Cd-SiNPs and CdCl₂ (1 - 50 μg/ml). Figure 3 describes a panel of representative and randomly selected microscopic fields of A549. The positivity of caspase-3 was absent and there were not appreciable morphology alterations of the A549 cells after SiNP exposure (Figure 3). Whereas, Cd-SiNP or CdCl₂ treatments showed dose-dependent caspase-3 activation, in particular already at the lowest dose tested of 1 μg/ml the activated form of caspase-3 was well visible as brilliant green spot into cytoplasm of the A549 cells (Figure 3). Cell morphology alterations characterized by cell shrinkage and chromatin condensation were observed at the highest dose (50 μg/ml) for both Cd-SiNP and CdCl₂ (Figure 3).

Figure 3. Immunofluorescence analysis of activated caspase-3 in A549 cells after 1-day treatment. Representative images of randomly selected microscopic fields of A549 cells marked with caspase-3 antibody after 24 h incubation with increasing concentrations of Cd-SiNPs, CdCl₂ and SiNPs. Activation of caspase-3 is indicated by green spots from the 1 μg/ml dose in both Cd-SiNPs and CdCl2 treatment groups, while there is no positivity in treated SiNP cells at all the doses tested. Scale bar: 100 μm

Figure 4. Intracellular GSH content in A549 cells after 1-day treatment. Glutathione measurement in A549 cells unexposed (control) and exposed to increasing concentrations (10 - 100 μg/ml) of Cd-SiNPs, CdCl₂, SiNPs after 24 h. GSH levels were affected by all tested materials. Data are mean ± SD of three separate experiments each carried out in six replicates. *Different from control 𝑃 < 0.05, Statistical analysis by ANOVA

3.2.3. Oxidative Stress: Intracellular Glutathione (GSH) Measurement

The intracellular GSH levels after 24 h exposure were affected by all test materials (SiNP, Cd-SiNP and CdCl₂). SiNP treatment showed dose-dependent depletion of GSH in A549 cells when compared to control with about 75% decrease at highest concentration investigated (100 μg/ml), and 55% decrease at the lowest dose of 10 μg/ml (Figure 4). The intracellular GSH reduction caused by Cd-SiNP and CdCl₂, at the doses from 10 to 100 μg/ml, ranged from 25 to 60%.

3.2.4. Clonogenic Assay

The proliferation ability and colony forming capacity of A549 cells were evaluated after the prolonged exposure (up to 10 days) to increasing concentrations (from 0.05 to 100 μg/ml) of Cd-SiNPs, SiNPs and CdCl2. The Figure 5a, b, c reports the results obtained from 0.05 to 10 μg/ml since doses ≥ 25 μg/ml of Cd-SiNPs and CdCl₂totally suppressed the cell growth. Clonogenic test is a non-colorimetric assay, to determine the proliferation ability of all cell types upon long-term exposure, it uses the ability of cells to form colonies (a colony being defined as at least 50 clones of one cell) when seeded at very low cellular concentrations, it identifies the cells that are destined to die or survive. SiNPs did not produce inhibition of the proliferative activity of cells after prolonged exposure to any dose tested; Figure 5c. A549 cells treated with doses ranging from 0.05 to 10 μg/ml of Cd-SiNPs and CdCl₂ presented few colonies (55 - 1% for both treatments) and a drastic reduced size (Figure 5a and b, respectively). At the higher concentrations from 25 to 100 μg/ml of either Cd-SiNPs or CdCl₂ there was a complete inhibition of the colony formation (data not shown).

Figure 5. Growth and cell proliferation of A549 cells after 10-day treatment. Representative images of randomly selected microscopic fields of the colonies formed after 10 consecutive days exposure to increasing concentrations (0.05 - 10 μg/ml) of Cd-SiNPs (a), CdCl₂ (b) and SiNPs (c). The formation of A549 colonies appears to be serious compromised by Cd-SiNP or CdCl₂ treatments, few colonies with reduced size compared to control were observed at the lower concentrations (0.05 - 10 μg/ml) of Cd-SiNPs and CdCl₂, while there was a total inhibition of the colonies at the higher tested doses (25 - 100 μg/ml) for both Cd-SiNPs and CdCl₂. (Scale bar: 600 μm). SiNP colonies showed similar patterns at the all tested doses to the control. Quantitative analysis of the colony number is shown for each treatment. Histograms show the number of colonies formed after 10 consecutive days Data are mean ± SD of three separate experiments. *Different from control 𝑃 < 0.05, Statistical analysis by ANOVA. Error bars: ± SD

3.3. In Vivo Results

The in vivo experiments performed as the second part of this work addressed the pulmonary effects of the compound types (SiNP, Cd-SiNP, and CdCl₂) in terms of apoptosis, morphological analysis and oxidative stress after exposure (1 and 7 days) by i.t. instillation.

Although differences exist in the dose rate and distribution of material delivered by instillation versus inhalation, however, the data base on pulmonary toxicity of various particulate materials indicates that the two modes of exposure yield qualitatively similar results for a variety of biological endpoints, including pulmonary inflammation, fibrosis, susceptibility to infection, allergic sensitization and lung cancer in rats after exposure to poorly soluble particle [43]. In particular for Cd, as discussed by Oberdörster [44,45,46], the pulmonary retention half-time of both inhaled and i.t. instilled CdCl2 resulted in similar Cd retention and similar clearance kinetics (i.e. 60-85 days) in rats. For the present study the i.t. was applied as a useful method to compare the lung effects in rats of a new material (i.e., counterpart-doped nanoparticles) against similar compounds (i.e. CdCl2) for which an extensive inhalation database is available [47]. Moreover, i.t. model may serve to a screening tool to determine the approximate dose range that may be appropriate for later inhalation studies as well as to address specific endpoints regarding the respiratory toxicity of nanomaterials [43].

3.3.1. Apoptotic Phenomena: TUNEL Staining and Ultrastructural Evaluation (Electron Microscopy)

Cell death as a consequence of SiNPs-, Cd-SiNPs- and CdCl₂-exposure was assessed using TUNEL staining, being this a distinctive marker of apoptosis (Figure 6A, B; and Table 1). Any presence of necrosis was detected, nonetheless paralleled by the presence of several apoptotic cells. The canonical apoptotic phenomenon, characterized by TUNEL-positivity, and ultrastructurally, by nuclear pyknosis, karyorrhexis, and apoptotic body formation (Figure 6A), increased significantly after all three types of treatment, more markedly at the early time point (1 day) and persisting until 7 days (lessening with time) (Figure 6B, Figure 7). The more marked Cd-SiNP-dependent effects were mainly observed in the alveolar epithelial cells, compared to the bronchiolar cells and the macrophages, with the enhancement displaying the following trend: Cd-SiNPs>CdCl₂>SiNPs.

Figure 6. Apoptosis detected by TUNEL staining after 1 and 7 days treatments. TUNEL staining (A) and Electron Microscopy (B) in lungs of treated animals (d1-g1 and d7-g7): Representative micrographs showing apoptosis, detected by TUNEL staining, 1 day (d1-g1) and 7 days (d7-g7) after i.t. exposure to CdCl₂ (e1 and e7), Cd-SiNPs (f1 and f7) and SiNPs (g1 and g7). (d1 and d7) present the lung parenchyma specimens in control animals. After all treatments, TUNEL positive cells, typically characterized by chromatin condensation, were detected both in stromal and epithelial areas (e1-g1 and e7-g7), with the tendency to lessening with time. Labeled pneumocytes and macrophages were observable, with the Cd-SiNPs instillation causing the more marked apoptotic effect. (a1-c1 and a7-c7): Electron microscopy images illustrating different stages of apoptotic cell death, 1 day and 7 days-post exposure, respectively. (a1 and a7) pyknotic nuclei in early karyorhexis; (b1 and b7) manifest karyorhexis; (c1 and c7) late apoptosis characterized by apoptotic bodies presence, along with the presence of type II pneumocytes (PII) filled by intracellular surfactant-storing lamellar bodies (c7). Light microscopy scale bar: 200 microm (d1-g1 and d7-g7). Electron Microscopy original magnification: (a1 and a7) x 4400; (b1, b7 and c1) x 7000; (c7) x 3000

Figure 7. TUNEL Labelling Index modifications at two time points. Histograms showing changes in TUNEL Labelling Index percentage of lung cells, caused by i.t. instillation of Cd-SiNPs versus CdCl₂ or SiNPs, investigated at 1 and 7 days-post-treatment. In all exposed groups, a significant increase of apoptotic cells was evidenced, more manifest 1 day post-exposure, displaying the tendency to lessening with time, showing the following trend: Cd-SiNPs > CdCl₂ > SiNPs. Data are expressed as mean ± SD, (*) indicates statistically significant differences (P < 0.05) compared to the respective control. Diverse letters (a-c) denote mean values that are statistically different at P < 0.05: comparison is between one time point versus the one immediately before of the same group

3.3.2. Morphological Analysis

After a single i.t. exposure to the different compounds (i.e. SiNPs, Cd-SiNPs and CdCl₂) the lung parenchyma, analyzed by both light (H&E staining) and electron microscopy, clearly showed patterns of injury, with different extent of intensity, characterized by collapsed alveoli, altered inflammatory areas, granuloma formation, thickening of alveolar septa, along with areas presenting bronchiolar epithelium exfoliation (Figure 8c). Noticeably, the presence of vacuolized and intracellular surfactant storing lamellar bodies of various shapes and sizes characterized several type II pneumocytes (Figure 8d). Parallely, several activated alveolar macrophages were observed at ultrastructural level, at which several cytoplasmic eterophagosomes, containing internalized material in degradation (Figure 8e). The presence of several activated Club cells was also detected in the bronchiolar epithelium, showing apical protrusion containing several electron-dense secretary granules (Figure 8f). These overall effects were observed acutely (after 1 day) and lasted until the 7th day, with Cd-SiNPs treatment producing the more marked effects compared to SiNPs and CdCl₂ groups.

Figure 8. Lung histology. Representative lung parenchyma specimens, investigated by both Light (H&E) and Electron microscopy (a-c and d-f, respectively), from control (a) and Cd-SiNPs-treated rats, 7 days after i.t. instillation (b-f). Selected structural alterations evidenced in pulmonary parenchyma of Cd-SiNPs treated rats are presented. (a) normal, physiological alveolar and bronchiolar morphology; (b) bronchus-associated lymphoid nodule adjacent to granulomatous formation (arrow and thin arrow, respectively) in both peribronchiolar and perivascular area; (c) collapsed alveoli characterized by wall thickening and micro hemorrhagic foci, along with bronchiolar distortion and epithelial cell desquamation (arrow); (d) edematous alveolar wall (arrow) and type II pneumocytes (PII) packed with vacuolized multi-vescicular bodies; (e) activated alveolar macrophage (AM), typically characterized by the presence of cytoplasmic phagosomes and residual bodies; (f) activated Club cells (Cc) detected in the apical region of bronchiolar epithelium. Light microscopy scale bar: 200 microm (a-c). (Electron Microscopy original magnification: (d-e) x 4400; (f) x 3000)

3.3.3. SOD1 and iNOS in Lung

The localization and distribution of immunopositivity for (Cu/Zn-SOD) SOD1 and iNOS, essentially involved in oxidative stress pathway, revealed an extensive spreading in bronchiolar and alveolar cells, as well as in the capillary component, evidencing the pulmonary reaction to the injury, observable starting at 7 days after i.t. exposure (Figure 9; Table 1), with Cd-SiNP and CdCl₂ treatments causing adverse effects of comparable extent. Administration of SiNPs did not significantly influence any of the two investigated molecules (Figure 9; Table 1).

Figure 9. SOD1 and iNOS expression in rat lung tissue. Immunocytochemical labeling for SOD1 (a-d) and iNOS (e-h), in lungs of control (a and e), CdCl₂- (b and f), SiNPs-Cd- (c and g), and SiNPs- treated rats (d and h). The SOD1 expression was evidenced after both CdCl₂ and Cd-SiNPs treatments, 7 days post-instillation (b and c, arrows). Primarily detected at alveolar level (i.e. macrophages). The iNOS labelling was mainly observed in clusters of the inflammatory cells (arrows), surrounding the alveolar capillary wall, after both CdCl₂ and Cd-SiNPs exposure (f and g, respectively). SiNP-treated rats showed immunostaining patterns of SOD1- and iNOS-expression comparable to those observed in controls (d and h, respectively). Scale bar: 200 microm (a-h)

Distinctly, numerous SOD1, immunopositive activated macrophages were detected particularly evident in collapsed areas, appearing heavily labelled (Figure 9), as also demonstrated by ultrastructural evaluation, evidencing the presence of several eterophagosomes, containing internalized material undergoing degradation process.

The strongest iNOS-immunopositivity was observed at capillary level, along with the presence of several immunopositive inflammatory cells, after both Cd-SiNPs as well as CdCl₂ (Figure 9).

The enhancement in SOD1 and iNOS immunolabelling, with different expression patterns in bronchiolar, alveolar and vascular epithelium, observed after either Cd-SiNPs or CdCl₂ treatment, is consistent with (i) the central role played by both the investigated molecules in pulmonary system, mainly in the development and progression of lung injury, and (ii) the established function of the respiratory epithelia as the first line of defence after insult, also in accordance to previous in vitro and in vivo findings related to cadmium toxicity.

3.3.4. F2-IsoPs in Plasma and Lung

In lungs total F2-IsoPs levels were not modified at the earlier time points (1-7 days) after Cd-SiNPs and CdCl₂ i.t. treatments (Figure 10). Whereas, previous our studies evidenced significantly increased levels of F2-IsoPs after 30 days in both groups (Cd-SiNPs and CdCl₂) [48]: F2- isoprostane enhancements were 43% and 56% and in Cd-SiNP and CdCl₂ groups, respectively, compared to controls (32.8±7.8 ng/g). Changes in plasma F2-IsoPs were already observable at 7 days (by 79.5% and 94.9% and in Cd-SiNP and CdCl₂ treatments, respectively) and increases were still present at 30 days post-exposure (95.1% and 112.7% and in Cd-SiNP and CdCl₂, respectively, when compared to control, 28 ± 8 pg/ml). The lung and plasma F2-IsoPs levels were not significantly different between Cd-SiNPs and CdCl₂ groups, at both 7 and 30 days post-exposure. SiNP treatment did not produce any significant changes of F2-IsoPs levels in both the lung tissue and the plasma samples at all time points considered (Table 1).

Figure 10. Total F2-IsoPs levels in lungs and free F2-IsoPs levels in plasma of treated animals. Both pulmonary and plasma F2-IsoPs levels have been evaluated at 1, 7 and 30 days after i.t. exposure to Cd-SiNPs, CdCl₂, SiNPs. Data are means ± SD. Differences between groups were evaluated using two-tailed t test and P value < 0.05 was considered statistically significant (*). (Modified from Coccini et al. [48])

4. Discussion

Potential risk associated with pulmonary absorption of engineered Cd-SiNP exposure was assessed by using a complementary approach including in vitro and in vivo experiments. The cytotoxicity of Cd-SiNPs compared to the effects produced by the “SiNPs” counterpart (not doped) or by CdCl₂ was investigated in human lung carcinoma A549 cells performing a battery of in vitro tests namely: (i) MTT-assay, (ii) calcein-AM/propidium-iodide staining, (iii) GSH assay, (iv) clonogenic (colony forming) assay. The apoptotic pathways were also investigated by immunocytochemistry in fluorescence microscopy. Cells were exposed to different test items (1 - 100 μg/ml corresponding to 5.45 - 545 μM) for 1 day or 10 days (0.05 - 10 μg/ml corresponding to 0.27 - 54.5 μM).

The in vivo pulmonary effects of Cd-SiNPs (1 mg/rat), SiNPs (600 μg/rat) and CdCl₂ (400 μg/rat) were also evaluated at 1, 7 and 30 days after i.t.. Comprehensive characterization of the pulmonary damage in terms of apoptosis and morphological lung evaluation by H&E and EM was performed. Lung damages in terms of oxidative stress induction were also considered, by evaluating F2-IsoPs, as well as SOD1 and iNOS enzymes, and parallely by assessing the validity of plasma F2-IsoPs as marker of pulmonary insult.

The most significant results obtained by in vitro investigations showed that Cd-SiNPs produced cytotoxic effects after short- (1 day) and long-term (up to 10 days) exposure characterized by:

- mitochondrial function impairment and induction of apoptosis starting at 1 μg/ml (5.45 μM),

- intracellular GSH depletion at 10 μg/ml (54.5 μM),

- membrane alterations at 25 μg/ml (136.3 μM),

- inhibition of cell growth and proliferation observable already at 0.05 μg/ml (0.27 μM) after prolonged exposure.

Similar cytotoxic profile was observed after CdCl₂ treatment. The magnitude of effects caused by Cd-SiNPs was more pronounced compared to that produced by CdCl₂. In vitro, SiNPs induced effects on GSH content only.

All three treatments induced intracellular GSH level decrease, suggesting oxidative stress. Since the GSH is known to react as a chemical chelator for Cd, a decrease of this compound is expected in Cd-treated cells [49]. Indeed, alteration (decrease or increase) of GSH levels is dose-dependent [50]. Gaubin et al. [51] demonstrated that low Cd concentrations, i.e. ranging from 1 to 10 μM (∼ 0.2 - 2 μg/ml), produced a significant GSH increase level after 24 h exposure in A549 cells. On the other hand, using 40 μM (∼8 μg/ml) of Cd, no effect on thiol content was reported in A549 cell after 24 h [52]. The latter is in agreement with our data since the effect on GSH level (decrease) was observed starting at dose ≥ 10 μg/ml. Similarly, Cd concentrations ranging from 25 to 100 μM (∼ 5 - 20 μg/ml) resulted in a decrease of cell GSH level [51]. GSH reduction was also observed in hamster lung cells after 24 h exposure to 20 μg/ml CdS quantum dot [53].

Comparatively with our in vivo results, although lung GSH level was not measured, apparently no effect was observed on oxidative stress markers (i.e. SOD and iNOS) 1 day post-exposure. Homeostatic mechanisms probably counteracted the early (24 h) insult.

In vivo results revealed early and persistent lung damage after i.t. instillation of Cd-SiNPs (1 mg/rat, equivalent to about 250 μg of Cd) in terms of enhanced apoptotic phenomena and altered lung parenchyma morphology (collapsed alveoli, granuloma formation, inflammatory areas, macrophage activation, bronchiolar epithelium exfoliation). These effects were detectable at the earliest time point (1 day), and persisted until the 7th day post administration. Similar pattern of toxic insult was also revealed after i.t. instillation of equivalent amount of CdCl₂ and SiNPs, although in less marked manner than Cd-SiNP treatment.

Additionally, Cd-SiNPs and CdCl₂ caused a delayed occurrence of oxidative stress, evidenced at day 7 post dosing and lasting until day 30, by increasing the pulmonary SOD1 and iNOS expression as well as F2-IsoPs levels at day 30. At plasma level, the F2-IsoPs levels were significantly modified starting at day 7 by both Cd-SiNPs and CdCl₂. Pulmonary F2-IsoPS changes were delayed in onset and were preceded by marked increase of F2-IsoPs levels in plasma suggesting that plasma F2-IsoPs may be a sensitive target for Cd-induced lipoperoxidation and early predictive indicators of later pulmonary oxidative insult.

SiNPs, differently from Cd-SiNP and CdCl₂, did not cause oxidative stress evaluated by these parameters.

The in vivo results showed a higher Cd-SiNPs reactivity than CdCl₂ and SiNPs in the lung tissue.

The proposed research provided several major achievements with regard to the validity of in vitro test methods, namely a broad based scientific approach to a battery of in vitro assays applicable to target organ (lung) toxicity testing:

1. The in vitro results (in pulmonary cell line) were found to be predictive for some specific (lung) effects evidenced by in vivo studies such as apoptotic phenomena and oxidative stress, making the in vivo findings even useful for in vitro test validation. Moreover, the in vivo experiments gave further specific tissue information related to morphological tissue (pulmonary) alteration.

2. Both the short and prolonged in vitro exposure experiments have been helpful for the time course planning and for addressing and completing the toxicological response characterization performed in the animal tissue.

3. The in vitro applied doses have also been worthy to address the first step of in vivo experiments with explorative dosages.

In vitro and in vivo results on Cd-SiNP toxicity profile suggest that the lung is a susceptible target tissue and, lung morphological alteration, apoptosis and oxidative stress the main noticeable phenomena.

Both in vitro and in vivo findings pointed out that Cd-SiNP exposure produces a complex and multicomponent insult leading to an exacerbated toxicity response compared to the toxic pattern caused by CdCl₂ and SiNP treatment.

Altogether the results of these investigations support the concept that multiple assays and an integrated testing strategy should be recommended to characterize toxicological response to NPs. Similar approach could be applied to other types of engineered nanomaterials that are capable of entering the body and expressing biological activity related to their nanostructure.

From the bulk of in vitro and in vivo studies, the obtained exacerbated toxic effects induced by Cd-SiNPs suggest a crucial role of the cadmium moiety in the biological response to Cd-SiNPs although it seems unlikely that the changes produced by Cd-SiNPs merely reflected the action of Cd ions released from NPs. Indeed, chemical experiments with Cd-SiNPs have demonstrated limited release of Cd ions from the NPs dispersed in medium culture or physiological solution: the maximum metal release being ca. 28 or 15 %, respectively, over a 10-day period.

The tendency to form aggregates and agglomerates of these doped NPs [31] may have contributed in triggering the described Cd-SiNP effects: the dynamic light scattering data demonstrated an agglomeration and aggregation extent of Cd-SiNPs (about 350 nm) greater than that measured for SiNPs (about 120 nm). Whether those agglomerated particles retain toxic properties of the individual nanoparticles or are capable of subsequently de-agglomerating is a critical question [54].

An additional hypothesis may be related to the “nano” dimension of the material investigated. In our experiments, the ‘‘nano’’ dimension may have facilitated the cell concentration and lung toxicity of the administered cadmium. A ‘‘Trojan horse’’-type mechanism involving silica nanoparticles as effective carriers for the cellular uptake of toxic metals has been described [55]. In the same type of lung cells using in this study, exposure to SiNPs doped with metals such as iron, manganese, cobalt, or titanium was shown to generate higher concentrations of reactive oxygen species and induce more severe oxidative stress compared to equivalent amounts of the respective metal ions [56]. In our study, incorporation of Cd into SiNPs may have increased the metal dose delivered to target cells although no specific data supporting this process are presently available.

With regard to SiNPs, no in vitro cytotoxic effects were observed for all concentrations tested and for all exposure times (both in acute or prolonged evaluation), with the only exception for the observed GSH depletion. On the other hand, no oxidative stress has been observed after in vivo exposure, although SiNPs were able to produce morphological changes and apoptosis in lung tissue.

Recent literature data are in accordance with our data: at doses below 100 μg/ml SiNPs penetrated A549 cells without causing toxic effects at the molecular and cellular levels [57]; at concentrations up to 200 μg/ml SiNPs induced low cytotoxicity [58], and generated oxidative stress reflected by reduction of GSH levels [16] or oxidant generation [59]. Other investigations, however, indicated A549 cell viability decreases after SiNPs exposure down to 100 μg/ml [16], as well as a pro-inflammatory response triggered by SiNPs without blocking cell proliferation or causing cell death in A549 cells [60]. Our previous in vivo studies indicated SiNPs responsible of injury pattern of lung parenchyma and apoptotic phenomena, inflammatory responses and fibrogenic reaction [31], although less markedly than Cd-SiNPs.

With respect to the i.t. instillation exposure, several studies have compared inhalation to i.t. instillation proving that i.t. inhalation exposure is a suitable procedure to study the pulmonary toxicity induced by different compounds (e.g. CdCl₂ [45], alpha-quartz, TiO₂ [61], quantum dots [62], multi-wall carbon nanotubes [63], fullerene [64] carbon black [65], TiO₂NPs [66], etc.). Inhalation (single low dose and repeated exposure scenarios) produces response trends similar to responses from single exposures (by i.t. instillation) although the latter are more robust. It is suggested that although, high dose rate methods can be used for quantitative ranking of NP hazards, these data caution against their use for quantitative risk assessment [66].

For instance, the relatively high doses i.t. instillation applied in the present in vivo study, able to induce lung injury [34, 35], seem to reflect effects observed even at lower doses as reported in other recent studies using various Cd compounds and size and various animal models. For example, a significant increase in lung injury was observed at QD (cadmium-selenide (CdSe)) either 5 or 12.5 μg/rat after a single i.t. instillation at different time points (1, 3, 5, 7, 14, and 28 days): injury parameters peaked at day 7 post-exposure and remained elevated throughout the 28 day-period [67]. CdCl₂ inhalation (0.2% Cd nebulised) for 15 min induced transient bronchial inflammation, increased bronchial reactivity, and increased MMP-9 activity in lung lavage fluid in the dogs [68]. In another study, the induction of pulmonary toxicity was also demonstrated following 4-h inhalation (nose-only) to soluble CdCl₂ particles at various sizes, i.e. 33, 170, 637 and 1495 nm, all at a target concentration of 1 mg/m³: the effects were depended on the amount of deposited material, thus depending on the initial (aerodynamic) particle size. A recent work using CdONP 240 μg/m³/mice (dose relevant that could be found in an occupational setting) given by nose-only exposure tube for 3 h/day for 3 days, produced pulmonary inflammation, cell injury and tissue remodeling [69].

5. Conclusions

In vitro experiments in pulmonary cells have provided effective means of screening and ranking the tested materials (Cd-SiNPs > CdCl₂ > SiNPs) using multiple toxicological endpoints (i.e. mitochondrial and membrane alterations, induction of apoptosis, inhibition of growth and proliferation, and intracellular GSH depletion). Consistently, the in vivo results have systemically characterized the tissue damage evidenced by lung parenchyma injury, apoptotic phenomena, and the occurrence pulmonary oxidative stress in rats. The in vivo targeted tests have complemented and addressed the in vitro findings to ensure the adequate evaluation of nanoparticle hazard potential, also in terms of time of appearance and persistence of the toxicological features on living organism.

The results from in vitro test approach successfully drive the in vivo exposure experiments (by transitioning from quantitative and mechanistic toxicity tests in human cells to qualitative animal tests); and vice versa in vivo toxicity lung evaluation, provided basic evidence for accepting the utility of the applied in vitro battery of tests (at least from a mechanistic point of view although not in term of critical doses). These observations are in line with the matter addressing the issue of the in vivo relevance of some in vitro assays for evaluating the toxicity of NPs [70,71], since in vivo data represent an essential criterion for accepting the utility of in vitro assays for NP toxicity evaluation.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper

Acknowledgements

This work was supported by Grants from Italian Ministries of Health, Research and Education, and CARIPLO Foundation (Rif. 2011-2096).

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