The main routes of NP entry into the body are inhalation, transdermal entry, and ingestion [13]. Absorption of NPs through the skin is rather minimal as skin represents an effective barrier compared to much more permeable gastrointestinal tract and lungs [14]. The lungs are the most effective primary gateway for NPs [15] therefore we used this pathway for NP exposure. The mice were placed in whole-body inhalation chambers where the concentration of NPs was 0.956 × 106 NPs/cm3. This unique system corresponds with real situations in how animals are exposed to NPs present in the air. Airborne NPs can come into contact with various parts of the body. Additionally, the animals take care of their fur so that oral exposure to inhaled NPs can be even higher than in humans. The estimated deposited dose over the 11 weeks inhalation period was 1.684 µg of PbO per gram of mouse body weight (Table 1).
Table 1
Characterization of generated PbO NPs.
Characterization of PbO NPs
|
PbO
|
Number concentration
|
0.956 × 106 NPs/cm3
|
Surface area
|
4.21 × 109 nm2/cm3
|
Mode
|
34.6 nm
|
Geometric mean diameter
|
29.7 nm
|
Geometric standard deviation
|
1.69
|
Mass concentration
|
149.3 µg PbO/m3
|
Estimated deposited dose (after 11w)
|
1.684 µg PbO/g
|
This value was calculated (Supplementary file 1) based on previously published methodology [16, 17, 18]. A surface area of the respiratory tract acquires increasing attention as an important parameter in assessing the toxicity of nanoparticles, and, recently, it has been presented as the most biologically relevant dose metric for NPs toxicity in the lung. Consequently, the deposition of NPs in the respiratory tract was studied to assess the toxicological impacts of NPs entering the lungs. The deposition of PbO NPs in the mouse respiratory tract was calculated using a multiple-path particle dosimetry (MPPD) model [19, 20] for monodisperse aerosol with a geometric mean diameter of 29.7 nm and mass concentration of 149.3 µg/m3 (Fig. 1, Table 1).
The surface area of PbO NPs deposited in the extrathoracic, tracheobronchial, and alveolar regions of the mouse respiratory tract is 20.4%, 9.76%, and 23.2%, respectively, in the lung. Further, the simulation of inhaled PbO NPs deposited in different parts of the human respiratory tract was performed (Fig. 1D). The deposition fractions were calculated by the International Commission on Radiological Protection (ICRP) deposition model for the extrathoracic, tracheobronchial, and alveolar region. The total surface area of generated PbO NPs (i.e., 4.21×109 nm2/cm3; ST) was calculated by scanning mobility particle sizer (SMPS) spectrometer software from a measured particle size distribution. The data show that the surface area of PbO NPs deposited in the lung alveolar region (SA; 34.1% of the total surface area of PbO NPs) is much higher than the surface area of NPs deposited in the extrathoracic (SET; 7.3%) and tracheobronchial (STB; 4.5%) regions. The sum of the surface area of the NPs deposited in the alveolar and tracheobronchial regions of the lungs results in the so-called lung-deposited surface area (LDSA). The LDSA (STB + A) corresponds to 38.5% of the total surface area of inhaled PbO NPs, while the sum of the surface area of the NPs deposited in the extrathoracic, tracheobronchial, and alveolar region forms 45.8% of the total surface area of inhaled PbO NPs.
The depositions calculated using the MPPD model for the extrathoracic, tracheobronchial, and alveolar regions of the mouse respiratory tract are different from those calculated with the ICRP model for the human respiratory tract, which is probably mainly due to different morphometry (i.e., mouse × human) and aerosol parameters (i.e., polydisperse aerosol in ICRP model × monodisperse aerosol in MPPD model) used in the given models.
The total bodyweight of mice was not changed after 11-week PbO NP inhalation; however, the lung weight coefficient significantly increased in PbO NP animals (p < 0.05, Fig. 1). The weight coefficients of organs were expressed as wet weight of the organ (g)/dead body weight (g) x 100. The weight coefficient of kidney was also increased in PbO NP animals (p < 0.05 in the case of right kidney, there was no significant difference for the left kidney). The other organ weight coefficients for liver and spleen were not significantly altered after PbO NP inhalation.
The highest level of Pb after subchronic PbO NP inhalation was found in the kidney and lung
The content of Pb was significantly increased after 11 weeks of PbO NP inhalation compared to the controls (Table 2) in all target organs studied here.
Table 2
Concentration of Pb in organs following 11 weeks of PbO NP inhalation.
|
|
ctr
|
PbO
|
lung
|
range
mean
SD
|
< LOD*
|
2463–4432
3393
733
|
liver
|
range
mean
SD
|
< LOD*
|
667–1281
937
222
|
kidney
|
range
mean
SD
|
< LOD*
|
2679–4592
3502
693
|
spleen
|
range
mean
SD
|
< LOD*
|
512–1914
973
551
|
The highest concentration of Pb was found in the kidney; a comparable content of lead was detected in the lungs. The liver and spleen contained similar mean concentration of lead that was about 3.5-fold lower than the Pb level in the kidney and lungs. Remarkably, the spleen displayed the highest differences in the content of Pb among the individual animals. In the control animals, the levels of Pb were always below the limit of detection (LOD) in all organs.
Inhalation of PbO NPs caused chronic inflammation in the lungs accompanied by massive cell infiltrates with abundant foamy macrophages
After 11-week PbO NP inhalation, histopathological analysis of the lung revealed numerous changes indicating severe lung tissue damage (Fig. 2, Table S1), similar to that found after a 6-week PbO NP inhalation experiment in our previous study [21].
Here, we prolonged the exposure time to 11 weeks to particularly focus on more profound chronic inflammatory tissue reaction. As the result of 11-week exposure, the lungs exhibited remodeling of tissue, bronchiolitis and alveolitis, atelectasis, hyperemia and dilated blood vessels, alveolar emphysema, thickened septa, increased number of cells in the interstitium, and occasionally hemorrhage. Siderophages were also found in lung tissue (Fig. 2). Moreover, inflammatory infiltrates were seen around bronchioles and blood vessels. As summarized in Table S1, the histopathological changes to lungs were all statistically significant (p < 0.001) when compared to air-inhaling controls. Severe morphological alterations of the lungs were also observed at ultrastructural level (Fig. 2). Necrotic bronchiolar cells, bronchioles with accumulated neutrophils and macrophages, alveoli with cellular debris and inflammatory cells, and damaged membranes of lung cells, completed the image of deviations in lung tissues.
Expectably, Green Trichrome staining confirmed collagen fibres only in the walls of the vessels and bronchioles in both control and PbO NP exposed animals. However, collagen fibres as well as other signs of fibrosis were absent in alveolar parenchyma after PbO NP exposure (Fig. 2).
As 11 days long inhalation have produced in lungs large inflammatory infiltrates with abundant macrophages (Table S2), we then focused on details of such inflammatory change. Neutrophilic granulocytes, macrophages, mast cells, and lymphocytes were the main groups of the immune cells found in PbO NP exposed animals. Lymphocytes were dominant cells present in lung infiltrates (Fig. 2). The number of neutrophils (visualized as MPO-positive cells) was increased in PbO NP exposed animals (Fig. 2J–L), and similarly to lymphocytes, they accumulated predominantly in lung infiltrates. Additionally, neutrophils were observed in the bronchioles and within alveolar spaces after PbO NP inhalation. The number of mastocytes (as visualised by Toluidine Blue staining) was not significantly altered after 11-week PbO NP inhalation (Fig. 2M–O). Clusters of plasma cells (mature B-cells producing immunoglobulins) were found in vicinity of blood vessels and bronchioles (Fig. 2R). High amounts of alveolar surfactant revealed by transmission electron microscopy (TEM) further implies serious damage to lung function caused by 11-week exposure to PbO NPs.
Besides unraveling subcellular damage, TEM also confirmed the presence of metal NPs in the lungs after PbO NP inhalation, with lead nanoparticles accumulating predominantly in the endosomes of alveolar epithelial cells I (Fig. 2S–T).
Increased numbers of foamy macrophages were associated with altered expression of phospholipase C genes
Next, we focused on macrophages and their signalling since these cells are instrumental for all three phases of immune and inflammatory response: initiation, maintenance, and resolution [22]. In the animals exposed to PbO NP, enlarged macrophages were accumulated in the alveolar spaces, bronchioles, and interalveolar septa, with this accumulation being statistically significant (p < 0.05) as demonstrated by quantification of CD68 (marker of macrophages) cell positivity (Fig. 3A–C, Table S2).
A macrophage-mediated inflammatory response is regulated by phospholipase C (PLC) signalling. Phospholipase C is a family of enzymes that hydrolyse phospholipids and are involved in intracellular and intercellular signal transduction. PLCβ signalling plays a role in the expression of pro-inflammatory chemokines. PLCβ1 directly regulates the expression of monocyte chemoattractant protein-1 (MCP-1 or CCL2) [9]. Here, the expression of PLCβ1 mRNA in the lungs after PbO NP inhalation was significantly downregulated (p < 0.05, Fig. 3D). Phospholipases PLCγ controls the maturation and function of B and T lymphocytes (PLCγ1 for T-cells and PLCγ2 for B-cells) [23]. Subchronic PbO NP inhalation induced a statistically significant upregulation of expression of PLCγ2 mRNA (p < 0.05, Fig. 3D). The effect of PLCγ2 upregulation on B-cells can explain the high accumulation of lymphocytes observed after PbO NP inhalation in lungs (Fig. 2C, E). Clusters of plasma cells (activated B-cells) were found by the transmission electron microscopy in the alveolar septa in PbO NP exposed animals (Fig. 2R). Phospholipase PLCδ1 has a different effect on macrophage function compared to the above mentioned PLC members, as it negatively regulates inflammatory response in macrophages [24]. In consonance with this fact, PLCδ1 mRNA was slightly (statistically non significantly) decreased in animals exposed to PbO NP (Fig. 3D).
A large number of macrophages exhibited the appearance of foamy cells with many cholesterol crystals and lipid droplets in their cytoplasm, as shown by TEM (Fig. 3). As lipid homeostasis plays a crucial role during the transformation of macrophages into foamy cells, we have further focused on possible changes in lipid metabolism. Macrophages have on their cytoplasmic membranes receptors that are responsible for cholesterol uptake represented by cluster of differentiation 36 (CD36) and scavenger receptor A1 (SR-A1), and receptors that are responsible for cholesterol efflux represented by ATP-binding cassette (ABC) transporters ABCA1, ABCG1, and scavenger receptor B1 (SR-B1) [25]. The levels of mRNAs of these receptors remained mostly unaffected in animals exposed to PbO NP. The only exception was statistically nonsignificant increase in mRNA of Abcg1. This suggests that other factors than receptor expression are responsible for cholesterol accumulation in lungs observed here, which will be necessary to follow in future.
Microvesicular steatosis and increased cholesterol uptake develop in the liver upon PbO NP inhalation
The liver represents the central organ of the main metabolic processes. Exposure to metal NPs can trigger toxic effects in the liver, as documented in our previous studies [26, 27]. Here, inhalation of PbO NP prolonged to 11 weeks caused major liver remodeling, focal dystrophy, hydropic degeneration, and vacuolization of hepatocytes (Fig. 4, Figure S1, Table S3).
Hepatocytes displayed pronounced anisocytosis and anisokaryosis. Hyperemia, sinusoid damage, and focal necrosis were also present. Animals exposed to PbO NPs contained in their liver parenchyma numerous macrophages, and necrotic foci with inflammatory leukocyte infiltrates, predominantly lymphocytes and neutrophils. Infiltration of immune cells was also observed in the portal areas. However, when histopathologically scored, the morphological alteration in liver parenchyma in animals exposed to PbO NP were not statistically significant in comparison to controls.
As visualized by Green Trichrome staining, the quantity of collagen fibers in the portal areas, walls of blood vessels, and bile ducts were about the same in the livers of control animals and animals exposed to PbO NP (Fig. 4D), documenting the absence of liver fibrosis.
Previously, we have observed liver steatosis in animals exposed to PbO NP for 6 weeks [7]. Here, subchronic 11-weeks long exposure to PbO NP also caused steatosis in their livers (Fig. 4G) with numerous lipid droplets scattered in cytosol of hepatocytes, as shown by TEM. Ito cells displayed large lipid vacuoles (Fig. 4H).
Next, we analyzed receptors responsible for cholesterol uptake (CD36 and SR-A1) and cholesterol efflux (ABCA1, ABCG1, and SR-B1) in the liver. In contrast to lungs, in the liver mRNA expression of scavenger receptor CD36 and transporter ABCA1 were significantly deregulated (p < 0.01 and p < 0.05, respectively, Fig. 4). The expression of other receptors remained unchanged.
Taken together, PbO NP inhalation caused both morphological and functional alterations to liver, including the imbalance of lipid metabolism.
Inhalation of PbO NPs caused alterations in the number of liver macrophages
Liver macrophages, called Kupffer cells, are resident cells localized along liver sinusoid endothelial cells. It was previously estimated that every 100 hepatocytes are accompanied by 20–40 macrophages [28]. Thus, Kupffer cells contribute to liver parenchyma as a major cellular component involved in the maintenance of hepatic and systemic homeostasis. We used immunohistochemical detection of CD68 to visualize Kupffer cells in the liver (Fig. 5A–E, Table S4).
The number of hepatic macrophages was significantly increased (p < 0.05) after 11-week inhalation of PbO NPs (Fig. 5D).
We also measured expression of PLC genes in livers. Upon the exposure to PbO NPs, the levels of mRNAs for PLCβ1 and PLCγ2 remained completely unchanged, while the level of PLCδ1 became modestly lowered (Fig. 5F).
Energy dispersive X-ray (EDX) analysis differentiated lead particles from particles of iron in hepatocytes
Hepatocytes are major liver cells that physiologically store iron, one of the biogenic metal elements. Ultrastructurally, the particles of iron can be seen in the hepatocyte cytoplasm in the form of individual local agglomerates of small primary particles [29]. As lead nanoparticles can also form agglomerates (size range approximately 40–50 nm) of primary particles of 0.4–0.5 nm in diameter, we decided for EDX analysis to identify the chemical composition of particles observed by TEM.
Agglomerates of particles were randomly distributed in hepatocyte cytoplasm and mitochondria. Within one selected mitochondrion with agglomerates of particles, we analysed two different region of interest (ROI) windows (with and without agglomerates) by EDX. Spectra were compared with a reference sample and analyzed using Oxford AZtec. The presence of Pb was confirmed by comparing the reference Spectrum 31 (Fig. 5, without NPs) and non-reference Spectrum 30 (Fig. 5, with NPs) by increased signal at a spectral position of Pb. Therefore, hepatocyte mitochondria contained agglomerates of lead nanoparticles (Fig. 5).
Subchronic PbO NP exposure caused only minor changes to the kidney parenchyma, but blood vessels contained large lipid droplets
Since Pb is mainly excreted by kidneys [30], the effects of inhalation of PbO NP to this organ was also studied. The concentration of Pb in the kidney was higher than in all organs studied here; however, only minor morphological changes were observed in the kidney after the 11-week PbO NP exposure (Fig. 6, Figure S2, Table S5), similar to the 6-week exposure [21].
In some animals exposed to PbO NP, the glomerular metaplasia (change of female-like flattened cells in glomerular parietal epithelium of Bowman´s capsule to male-like cuboidal cells) was seen, dilatation of proximal tubules was observed in one animal, and mild inflammatory perivascular cell infiltrates were irregularly located in kidney cortex. The kidney medulla did not exhibit any pathological features. The amount of collagen fibers visualized by Green Trichrome in PbO NP inhaling animal was about the same as in the controls (Fig. 6).
At the ultrastructural level, the glomerular and tubular parts of nephrons were without alterations. The kidney filtration barrier exhibited a characteristic physiological appearance. Interestingly, large lipid vacuoles were present in some kidney blood vessels (Fig. 6). The renal tubules closely attached to such vessels were compressed and displayed damage to their epithelial lining. Accumulations of nanoparticles were seen in the epithelial cells of proximal tubules (Fig. 6).
Next, we analyzed the expression of receptors CD36, SR-A1, ABCA1, ABCG1, and SR-B1, similar to other target organs. In the kidney, only expression of ABCA1 mRNA was significantly lowered (p < 0.01, Fig. 6), the expression of other receptors remained at about the same level as in the controls. In both glomerular and tubular compartments of kidney the CD68-positive macrophages were scarce, with no difference between the control and PbO NP inhaling animals.
The expression of PLCβ1, PLCγ2, and PLCδ1 mRNAs in the kidney did not exhibit any significant changes upon the exposure of animals to PbO NPs.
The exposure to PbO NP did not lead to alterations in spleen macrophage distribution and morphology
After the 11-week PbO NP inhalation, the concentration of Pb in the spleen was at the same level as in the liver. However, histopathological analysis of the spleens of PbO NP inhaling animals did not unravel any significant changes (Fig. 7). The proportion of red and white pulp was about the same in both PbO NP-exposed and control animals. CD68-positive cells were detected predominantly in the white pulp where macrophages typically reside. Collagen fibers were rare in splenic parenchyma in both PbO NP exposed and control animals. The only change observed in animals exposed to PbO NPs was increased number of megakaryoblasts and megakaryocytes in splenic parenchyma.