A Histological Study on the Acute Effect of Zinc Oxide Nanoparticles Administered by Different Routes on Albino Rat Lung.

Introduction and Aim of the Work: Zinc oxide nanoparticles (ZnO NPs) are considered the most frequently utilized NPs, so the potential for human exposure has increased tremendously. Hence, the study is aimed to compare the histopathological effects of high and low doses of ZnO NPs administered intranasally or intravenously on lung tissue of adult rat's male albino. Materials and
Methods: Thirty-five male Wistar rats were divided into Group I; control group, Group II (intranasal administered group) was subdivided into Subgroup IIA and IIB, in which the animals were injected with 4 and 30 mg/kg of ZnO NPs, respectively. Group III (intravenous administered group) was subdivided into two subgroups with the same doses as Group II. Blood samples were collected after 24 h for estimating serum level of lactate dehydrogenase. Rat lungs were processed for histological, immunohistochemical, and ultrastructural analysis.
Results: ZnO NPs caused thickening of interalveolar septa. Extravasated red blood cells were noticed in the alveolar lumen and in some bronchioles. Many dilated blood vessels exhibited focal disruption and focal thickening of their wall. Collagenous fibers were deposited in the interalveolar septa and the walls of bronchi. Tumor necrosis factor-alpha immune reactivity was significantly increased. These findings increased on dose increase, mainly in the intranasal administered group when compared with the intravenous group.
Conclusion: ZnO NPs administration caused toxic effects on the histological structure of albino rat lung. These effects were route and dose-dependent, being more obvious after intranasal administration. Copyright:

INTRODUCTION

The nanoparticles (NPs) are engineered compounds with size ranging between 1 and 100 nm.[1] The large surface area: volume ratio of NPs led to an alteration in their biological activity compared to the parent bulk materials.[2] By modifying their size, surface properties, and shape NPs can be used in an application-specific manner.[3] Zinc oxide NPs (ZnO NPs) are considered one of the commonly utilized NPs. They enter in many industries and biomedicines such as personal hygiene products, textiles, electronics, and cosmetics.[4] Furthermore, they are important component of sunscreens and moisturizers,[5] food industries and in packaging.[6] Furthermore, their anticancer effect was documented.[7]

These ZnO NPs could access the body through skin topical application, lung inhalation, food consumption, and intra venous injection.[8] It was reported that the major route for entry of ZnO NPs is the respiratory system.[9] Nevertheless, the possibility of intravenous injection of ZnO NPS has been elevated as a carrier for different chemotherapeutic.[10] Due to their wide surface area and small size, they could easily pass the physiological barriers and be widely distributed in circulation.[11] It is, therefore, necessary to assess their toxic effect to avoid their potential adverse effect.[12]

This study was designed to compare potential histopathological effects of high and low doses of ZnO NPs, either administered intranasally or intravenously on the histological structure of the rat lung.

MATERIALS AND METHODS

Preparation of zinc oxide nanoparticles

ZnO NPs were bought from “NanoTech Egypt for Photo-Electronics,” October 6, city, Giza, as white powder, of size = 20 ± 10 nm (surface area 15–25 m2/g and purity >99%). They were dissolved in 0.4 ml phosphate-buffered saline (PBS) with 5% bovine serum albumin (BSA). The ZnO NPs suspension was then sonicated for 10 min.[13]

Characterization of zinc oxide nanoparticles

Transmission electron microscope (TEM) (JEM-1200 EX II Electron Microscope, Tokyo, Japan) was used in evaluating the size, shape, and aggregation state of ZnO NPs. The NPs were grinded and diluted in PBS, 5% bovine serum was added to form a suspension which was deposited on copper grids coated with carbon. The grids were left to dry at ambient temperature before examination by the TEM.[14]

Animals

Thirty-five adult male Wistar rats of average 250 g were bought from and housed in Medical Ain Shams Research Institution, Faculty of Medicine, Ain Shams University, Cairo, Egypt. All animal procedures were performed in compliance with the general guidelines for the care and use of laboratory animals and approved by the animal ethical committee at the Faculty of Medicine, Ain Shams University. The animals were kept in plastic cages and mesh wire protections and were given rat water and chow ad libitum. The animals were exposed to 12 h of artificial light and 12 h of darkness throughout the experiment.

Experimental groups

In the present study, the rats were separated into three main groups:

Group I (control group): It included 15 rats, which were further subdivided into Subgroup IA: (n = 5), which were left without interference. Subgroup IB: (n = 5) which received single intranasal administration of 0.4 ml PBS with 5% BSA. Subgroup IC: (n = 5) which received single intravenous injection of 0.4 ml PBS with 5% BSA

Group II (intranasal administered group): It included 10 rats, which were further subdivided equally into: Subgroup IIA (low-dose intranasal subgroup): (n = 5) which received 4 mg/kg ZnO NPs intranasally as a single dose, dissolved in 0.4 ml PBS with 5% BSA.[15] Subgroup IIB (high-dose intranasal subgroup): (n = 5) which received 30 mg/kg ZnO NPs intranasally as a single dose, dissolved in 0.4 ml PBS with 5% BSA.[16]

Group III (intravenous administered group): It included 10 rats, which were further subdivided equally into: Subgroup IIA (Low-dose intravenous subgroup): (n = 5) which received 4 mg/kg ZnO NPs intravenously in tail vein as a single dose, dissolved in 0.4 ml PBS with 5% BSA.[15] Subgroup IIB (High-dose intravenous subgroup): (n = 5), which received 30 mg/kg ZnO NPs intravenously as a single dose in tail vein, dissolved in 0.4 ml PBS with 5% BSA.[16]

The experimental animals of all groups were sacrificed one day after ZnO NPs administration by cervical dislocation after ether inhalation. Just before sacrificing the animals, blood samples were aspirated from the aorta. The lungs were then dissected out and the bodies of the dead animals were disposed by using the incinerator. The lung specimens were processed for histological and ultrastructural examination.

Biochemical analysis of serum level of lactate dehydrogenase

Blood specimens were taken from the aorta and sent to the clinical pathology department, Faculty of Medicine, Ain Shams University. The samples were centrifuged to separate the serum fractions, kept in clean bottles and frozen at −20°C till processed. The released lactate dehydrogenase (LDH) was determined with commercially available LDH-Kits (CytoTox 96 Kit).[17]

Histological study

The right lung of each animal was fixed in 10% neutral buffered formaldehyde, dehydrated, cleared and impregnated in soft paraffin, then embedded in paraffin blocks. Serial sections were taken of −5 μm thick and processed for staining by hematoxylin and eosin stain and Masson's trichrome stain.[18]

Immunohistochemical study

Immunohistochemical staining for the inflammatory marker tumor necrosis factor-alpha (TNF-α) was done. It was purchased from LABVISION USA as monoclonal mouse anti-human TNF-α antibody (dilution 1:500) product number: (MAB510). The site of antibody binding was visualized by 3,3’-diaminobenzidine staining, which appeared as dark-brown cytoplasmic discoloration. Negative control slide was prepared in which PBS was used instead of the primary antibody.[19]

Transmission electron microscopic study

The left lungs of each rat in all subgroups were divided into 1 mm3 pieces and fixed in 1.5% glutaraldehyde to be further processed into epoxy resin-filled capsules. Ultrathin sections of 60–80 nm thick were cut, mounted on copper grids, and stained with the metal stains uranyl acetate and lead citrate[18] to be examined and photographed by TEM (JEM-1200 EX II Electron Microscope, Tokyo, Japan) in the regional center for mycology and biotechnology, Ain-Shams University.

Morphometric study and statistical analysis

Statistical analysis for the serum level of LDH of all rats in each subgroup was done

Five nonoverlapping different fields were studied in all sections of the lung of all rats in each subgroup to measure the following parameters:

Mean area percentage of collagen fibers in Masson's trichrome-stained sections (×40)

Mean number of TNF-α immunopositive cells in immunohistochemically stained sections (×40).

All measurements were performed by the image analyzer using Leica Q win software installed on a Dell PC (Texas, USA). The PC was connected to a microscope (Leica microsystem, Ehrenburg, Switzerland). Statistical analysis was performed using the software program SPSS, version 20 (IBM corporation, Armwork, North Castle, Westchesten Country, New York, USA). The statistical difference among groups for each parameter was determined using two-way analysis of variance followed by post hoc test with the least significance difference. For comparison between more than two groups, P ≤ 0.05 was considered statistically significant.

RESULTS

Serum lactate dehydrogenase level

Serum LDH level was significantly increased in all examined subgroups; IIA, IIB, IIIA, and IIIB (P < 0.05) as compared to the control group.

Comparing the low-dose subgroups, a statistically significant decrease of serum LDH level was detected on intravenous ZnO NPs administration in subgroup IIIA as compared to the intranasal administration in subgroup IIA (P < 0.05). Likewise, the high-dose subgroup showed a significant decrease of the serum LDH level after intravenous ZnO NPs administration in subgroup IIIB as compared to the intranasal administration in subgroup IIB (P < 0.05).

Comparing both subgroups of the intranasally administered ZnO NPs Group (II), a significant increase of serum LDH level was detected in the subgroup IIB as compared to the low-dose subgroup IIA (P < 0.05). Same finding was recorded when comparing subgroups of the intravenously administered ZnO NPs Group (III), as a significant increase of serum LDH level was detected in subgroup IIIB in comparison with subgroup IIIA (P < 0.05) [Table 1].

Table 1

Serum lactate dehydrogenase level in different groups

	Group 1	Subgroup IIA	Subgroup IIB	Subgroup IIIA	Subgroup IIIB
Serum LDH level	193.25±29.14	903.5±71.57a	1406±128.41a,b	497.75±58.66a,b	1152.5±28.41a,c,d

Values are presented as mean±SD. a Compared to Group I statistically significant (Control group) (P<0.05), b Compared to Group IIA statistically significant (low-dose intranasal subgroup) (P<0.05), c Compared to Group IIB statistically significant (high-dose intranasal subgroup) (P<0.05), d Compared to Group IIIA statistically significant (low-dose intravenous). LDH: Lactate dehydrogenase, SD: Standard deviation

Examination of NPs using TEM revealed aggregates of spheroid tiny electron-dense particles of a relatively similar size range of 15.3–34.8 nm [Figure 1].

Figure 1

A transmission electron micrograph showing variable-sized electron-dense aggregations of ZnO NPs with a size range of 15.3–34.8 nm (TEM, ×30,000). TEM: Transmission electron microscope

H and E-stained sections

No structural differences were observed in all subgroups of the control animals of Group (I). The lung appeared having sponge-like architecture. The alveolar wall was lined by the alveolar epithelium: Type I and II pneumocytes. Type I pneumocytes formed most of the alveolar epithelium, appearing flat with flattened nuclei. Type II pneumocytes were scattered cuboidal cells having round nuclei and were bulging into the alveolar lumina. Thin interalveolar septa were separating alveolar spaces. Simple cubical or low columnar epithelium was seen lined the bronchioles with visible bulging dome-shaped club cells in between [Figure 2a].

Figure 2

(a) Control group (i), showing type I pneumocytes (↑), type II pneumocytes (▲) lining the alveoli. Notice the simple columnar epithelium (↑↑) with intervening club cells (Δ) lining the small bronchiole (b). (b) Subgroup IIA, showing thickened interalveolar septa (blue↑) including dilated thickened blood vessel (↑) with disrupted endothelium (Δ). Mononuclear cell infiltrations (▲) and hyaline acidophilic exudate can be seen (*). (c and d) Subgroup IIB, showing thickened interalveolar septa (blue▲) by extravasated blood (↑) together with hemosiderin granules (▲) with collapsed adjacent alveoli (*). A pulmonary blood vessel appears dilated and congested (v) with disrupted wall (#). The bronchiolar wall appears infiltrated with inflammatory cells (↑). (e) Subgroup IIIA, showing few mononuclear cells infiltrating the mildly thickened septa (↑). Notice the presence of homogenous acidophilic exudate in the interalveolar septa (*). (f) Subgroup IIIB, showing exfoliation of epithelial cells including club cells with pyknotic nuclei (▲), extravasated RBCs and acidophilic exudate inside the bronchiolar lumen (*). Notice cytoplasmic vacuolations of smooth muscle fibers of the bronchiolar wall (↑) and the mononuclear cellular infiltration (blue▲) (H and E, ×400; c: H and E, ×100)

In subgroup IIA, few focal areas of apparently thickened interalveolar septa were seen infiltrated with mononuclear cells. Many dilated blood vessels exhibited focal disruption and focal thickening of their tunica media together with mononuclear cellular infiltration. Furthermore, acidophilic hyaline exudate was seen filling the blood vessels lumina [Figure 2b].

On examination of subgroup IIB, apparent thickening of many interalveolar septa was demonstrated in comparison with Group I and to subgroup IIA. These thickened septa, as well as the bronchiolar wall, were seen heavily infiltrated by mononuclear cells. Apparent thickening of most of the blood vessels’ wall was observed as compared to Group I and to subgroup IIA. Focal disruption of the blood vessels’ endothelium and muscle fibers of the tunica media was noticed. Some of these blood vessels were seen congested [Figure 2c and d].

On the other hand, subgroup IIIA showed minimal thickening of some interalveolar septa in a few focal parts could be detected. Mild infiltration of few interalveolar septa with few mononuclear cells together with homogenous acidophilic exudate could be seen [Figure 2e] (you mean as compared to control).

In subgroup IIIB, exfoliated epithelial cells were seen occupying the lumen of many bronchioles, with the appearance of many club cells with pyknotic nuclei. In contrast to subgroup (IIB), some bronchioles demonstrated extravasated red blood cells (RBCs) and acidophilic exudate in their lumen. Cytoplasmic vacuolations were also seen in the smooth muscle fibers of the bronchiolar wall in some areas. Mononuclear cellular infiltrated the bronchiolar walls [Figure 2f].

Masson's trichrome stained sections (histogram 1)

Examination of Group I sections revealed the presence of few collagen fibers in the interalveolar septa and in the walls of bronchioles [Figure 3a]. The mean area percentage of collagen fibers was measured as 1.15 ± 0.59.

Figure 3

(a) Control group (I); (b): Subgroup IIA; (c): Subgroup IIB; (d): Subgroup IIIA; (e): Subgroup IIIB, showing the collagen fibers distribution in the inter alveolar septa (▲), in the walls of the bronchiolar passage (blue↑), and in the wall of a bronchus (↑) (Masson’s trichrome, ×400). (f) Histogram 1 showing mean area percentage of collagen fibers measured in Masson's trichrome stained sections. aStatistically significant compared to Group I (P < 0.05), bStatistically significant compared to Group IIA (P < 0.05), cStatistically significant compared to Group IIB (P < 0.05), dStatistically significant compared to Group IIIA (P < 0.05)

Subgroup IIA showed few collagenous fibers deposition in the interstitial tissue between the alveoli and in the adventitia of the bronchioles [Figure 3b]. The mean area % of collagen fiber was significantly increased (P < 0.05) as compared to Group I, measuring 8.36 ± 2.5.

Moreover, subgroup IIB demonstrated an apparent increase in the content of collagen fibers in the interalveolar septa as compared to Group I and subgroup IIA [Figure 3c]. The mean area % of collagen fiber was significantly increased (P < 0.05) as compared to Group I and subgroup IIA, measuring 21.08 ± 4.97.

On the other hand, subgroup IIIA exhibited minimal collagenous fibers deposition in the interstitium in between alveoli and the walls of the bronchioles [Figure 3d]. The mean area % of collagen fiber was detected as 4.67 ± 1.41, which was significantly decreased as compared to subgroup IIA.

Subgroup IIIB revealed an apparent mild increase in the content of collagen fibers in the walls of bronchi and in the interalveolar septa in focal areas as compared to those of the control group. Nevertheless, they were apparently less than that of subgroup (IIB). Some collagen fibers were seen, extending to alveolar spaces [Figure 3e]. The mean area percentage of collagen fibers was 18.26 ± 4.24. That was significantly decreased as compared to subgroup IIB. Although, it was significantly increased as compared to Group I and subgroup IIIA [Figure 3f].

Immunohistochemical analysis of tumor necrosis factor-alpha (histogram 2)

The control group showed a minimal positive immune reaction in cells of the interalveolar septa [Figure 4a]. The mean number of TNF-α-positive cells was 2.2 ± 1.3.

Figure 4

(a) Control group (I); (b) Subgroup IIA; (c) Subgroup IIB; (d) Subgroup IIIA; (e) Subgroup IIIB, showing representative immunoperoxidase images for distribution of TNF-α in the rat lung. Dark-brown staining indicates sites where TNF-α present in the inflammatory cells of the interalveolar septa (↑) (avidin-biotin-peroxidase for TNF-α, ×400). (f) Histogram 2 showing mean number of immune positive TNF-α cells. aStatistically significant compared to Group I (P < 0.05), bStatistically significant compared to Group IIA (P < 0.05), cStatistically significant compared to Group IIB (P < 0.05), dStatistically significant compared to Group IIIA (P < 0.05). TNF-α: Tumor necrosis factor-alpha

Subgroup IIA showed mild positive cytoplasmic immune reaction in mononuclear cells infiltrating the interalveolar septa [Figure 4b]. The mean number of TNF-α-positive cells was 10.4 ± 1.82, which was significantly increased as compared to Group I.

Subgroup IIB demonstrated apparent increase in the TNF-α immune reactivity as compared to that of Group I and subgroup IIA. This was reflected by the presence of numerous immune positive mononuclear cells exhibiting brownish cytoplasmic granules [Figure 4c]. The mean number of TNF-α-positive cells was 24.8 ± 3.35, which was significantly increased (P < 0.05) as compared to Group I and subgroup IIA.

On the other hand, subgroup IIIA revealed less brownish cytoplasmic immune reaction in the alveolar macrophages as compared to those of the subgroup IIA [Figure 4d]. The mean number of TNF-α positive cells was significantly decreased as compared to subgroup IIA, measuring 5.2 ± 1.3.

In addition, subgroup IIIB demonstrated moderate positive brown immune cytoplasmic reaction, mainly in the mononuclear cells infiltrating the interalveolar septa. This was apparently increased when compared to that of the control group and both subgroups IIA and IIIA, however, apparently less than that of subgroup IIB [Figure 4e]. The mean number of TNF-α positive cells was 16.6 ± 2.3. This value was significantly decreased as compared to subgroup IIB. Yet, it was significantly increased as compared to Group I and subgroup IIIA [Figure 4f].

Ultrastructural examination of lung

Group I showed type I pneumocytes with their flat, slightly elongated euchromatic nuclei and few organelles in their attenuated cytoplasm. Type II pneumocytes could be easily recognized with their large round euchromatic nuclei and characteristic rounded lamellar bodies. Lysosomes could be seen inside their cytoplasm, and short microvilli were observed protruding from their surface [Figure 5].

Figure 5

A TEM of control group (I), showing type I pneumocyte with elongated euchromatic nucleus (N). Type II pneumocyte can be seen with round large euchromatic nucleus (n), lamellar bodies (▲) and lysosomes (red↑). Notice the presence of short microvilli (↑) (TEM, ×1500). TEM: Transmission electron microscope

Subgroup IIA showed type II pneumocytes with few dissolved lamellar bodies. Alveolar macrophages were seen with the large eccentric hyperchromatic nucleus and many lysosomes. Moderately electron-dense material was noticed in the interalveolar septum [Figure 6].

Figure 6

A TEM micrograph of subgroup IIA, showing a macrophage (M) with its large eccentric nucleus, in the alveolar space with many lysosomes (red↑) and vacuoles (v). Notice partially dissolved lamellar bodies of pneumocyte type II (blue↑) (TEM, ×1500). TEM: Transmission electron microscope

Subgroup IIB showed multiple cytoplasmic vacuolation of partially dissolved lamellar bodies of pneumocyte type II. Extravasated RBCs were seen obliterating the capillaries in the lung interstitium. Alveolar macrophages were noticed in the alveolar space and in the septa in between alveoli. They appeared with large eccentric hyperchromatic nucleus and many lysosomes [Figure 7a and b].

Figure 7

TEM micrographs of subgroup IIB, (a): PII with partially dissolved lamellar bodies can be seen (Δ). Notice the RBCs in the blood capillary (r). (b) Infiltration of the alveolar space (S) with macrophages (M) having large eccentric nucleus (N) and many lysosomes (red ↑) can be observed (TEM, ×1500). TEM: Transmission electron microscope, RBCs: Red blood cells, PII: Pneumocyte type II

In subgroup IIIA, few pneumocyte type II appeared with dissolved lamellar bodies. There was the infiltration of the interstitium with eosinophils with their bilobed nuclei and oval granules with electron dense core [Figure 8].

Figure 8

A TEM micrograph of subgroup IIIA, showing infiltration of the interstitium with eosinophil (E), having bilobed nucleus (N) and containing its specific cytoplasmic granules (G). Notice the dissolved lamellar bodies of pneumocyte type II (↑) (TEM, ×1500). TEM: Transmission electron microscope

Subgroup IIIB showed the appearance of some type I and type II pneumocytes having nuclei with irregular nuclear envelopes. Irregular chromatin distribution was seen in the nuclei of pneumocyte type II, together with partial dissolved lamellar bodies [Figure 9a]. Interalveolar septa showed infiltration of the interstitium with eosinophils exhibiting cytoplasmic specific granules with electron-dense core. Macrophages were also seen with many lysosomes and phagocytic vacuoles [Figure 9b].

Figure 9

TEM micrographs of subgroup IIIB, (a) irregular nucleus of PI. PII appears irregular with abnormal chromatin distribution. Partially dissolved lamellar body can be seen in pneumocyte type II (red↑). (b) Infiltration of the interstitium with an eosinophil (E) appears with specific electron-dense granules with dense core (G). A macrophage (M) can be seen with multiple phagocytic vesicles (TEM, ×1500). TEM: Transmission electron microscope, PI: Pneumocyte type I, PII: Pneumocyte type II

DISCUSSION

The current study evaluated the histological and ultrastructural changes in rat lung induced by intranasal and intravenous injection of ZnO NPs in rats. Wide variation in NPs toxicity was reported, and not only depends on their size and dose but also on the route of administration and duration of exposure.[20] Hence, in the present study, low and high doses of ZnO NPs (4 and 30 mg/kg, respectively) were used once intranasally and intravenously, aiming to detect the acute consequence of increasing dose ZnO NPs administration by dissimilar routes on the rat lung structure.

Examination of ZnO NPs by TEM in the present study showed their size ranging between 15 nm and 34 nm. The smaller-sized NPs exhibited greater particle solubility than the particles of larger surface area. The small size of NPs might be a cause of their high surface area. Consequently, this might increase their binding to serum proteins and promote their surface receptors recognition, leading to more tissue harm than larger-sized NPs.[21]

In the current work, the LDH levels were increased in all subgroups compared to Group I. However, there was a significant increase in the high-dose subgroups IIB and IIIB as compared with the low-dose subgroups IIA and IIIA. ZnO NPs was reported to cause elevation of the serum level of LDH in the rat after repeated oral administration.[22] It was also reported to be significantly increased in mice after exposure to ZnO NPs by 24 h, proposing that ZnO NPs caused acute lung cytotoxicity.[23] The acute increase in the level of LDH proposed that cell death was taking place in the lungs as early as 24 h after ZnO NP exposure.

In the present study, the interalveolar septa with apparent thickening by inflammatory cells infiltration were demonstrated in all experimental subgroups compared to Group I. However, there was increased thickening after high-dose administration in subgroups IIB and IIIB as compared with low dose in subgroups IIA and IIIA (intranasal and intravenous routes, respectively). This thickening led to the narrowing of some air spaces and compensatory widening of others. Moreover, extravasation of RBCs in lung alveolar spaces and alveolar septa was shown in the current study mainly in the intranasal group and in the high-dose subgroup (IIIB) of the intravenous group. Apparent thickening of most of the blood vessels’ and focal disruption of the blood vessels’ endothelium wall was detected as compared to Group I.

Going with these findings, ZnO NPs were reported to persuade an imbalance between oxidant and antioxidant systems in which there is an increase in the oxidant ones that cause damage.[24] The reactive oxygen species can disturb mitochondrial function and may also cause variations in the expression of genes that are involved in inflammation and apoptosis. An imbalance between antioxidant defense and free radical's production causes oxidative stress resulting in cell damage, and it is also very important in the pathogenesis of some diseases.[25]

In the present study, exfoliated epithelial cells were seen in high-dose intravenous subgroup (IIIB) occupying the lumen of many bronchioles. The epithelial shedding could be a consequence of some toxic inflammatory mediators. Furthermore, the increased epithelial fragility and shedding might be due to the weak attachment of the epithelial cells to the basement membrane.[26]

In the present experiment, macrophages were observed by TEM. After NPs engulfing by macrophages, they induce an inflammatory reaction and stimulate the production of interleukin (IL)-1β, IL-18, and TNF-α.[27] This can explain the presence of numerous mononuclear cells, including eosinophils and macrophages which were noticed by TEM in both subgroups of the intranasal group, and in the high-dose intravenous subgroup (IIIB) of the present work. Eotaxin and IL-13 were reported to be secreted in rats exposed to ZnO NPs and were suggested to be key mediators of eosinophil recruitment. IL-13 was stated to be tangled in the regulation of eosinophil infiltration and immunoglobulin E synthesis. Therefore, IL-13 activated by ZnO NPs might play a vital role in the development of the histopathological effects that demonstrated upon ZnO NPs exposure.[28]

In the present study, examination of Masson's trichrome-stained sections showed the presence of collagen fibers in the alveolar and bronchial walls. There was a significant increase in the area percentage of collagen fibers in all experimental subgroups as compared to Group I. However, there was a significant increase after high-dose administration in subgroups IIB and IIIB as compared with low dose in subgroups. TNF-α was documented to increase the manufacture of transforming growth factor-β1, which was the major stimulant of fibroblasts to secrete collagen fibers. Meanwhile, IL-1 β was also reported to increase the expression of platelet-derived growth factor (PDGF) and its receptors on the fibroblasts of the lungs. Therefore, the synchronized secretion of PDGF and its receptors might play a role in attracting fibroblasts from the interalveolar connective tissues and stimulate the proliferation of myofibroblasts. These cells began to form and deposit immature collagen fibers within the lung interstitium.[29]

Immunohistochemical stained sections for TNF-α in the present work revealed a significant increase in the number of immune positive inflammatory cells infiltrating the interalveolar septa. This was detected in all experimental subgroups as compared with that of Group I. Meanwhile, they were significantly increased after high-dose administration in subgroups IIB and IIIB as compared with low dose in subgroups. TNF-α production was increased in bronchoalveolar lavage samples of mice after 10 and 20 μg ZnO NPs exposures for the 24-h follow-up experiment.[23] The TNF-α is considered an early marker of inflammation. It is produced by many cells, such as activated macrophages, endothelial cells, and epithelial cells, and it causes recruitment and activation of neutrophils.[30] Similarly, intratracheal instillation of ZnO NP in rat increased inflammatory cells and many cytokine levels as TNF-α and IL-6 in the bronchoalveolar fluid at 24 h after administration of ZnO NP. Consequently, TNF-α was suggested to cause ZnO NP-induced acute inflammatory reactions with enrollment of neutrophils, monocytes, and macrophages.[31] Hence, it can be suggested that ZnO NPs might cause activation of the immune cells to secrete cytokines that result in inflammation. The ZnO NPs-induced TNF-α expression was previously demonstrated to be mediated through the ROS-ERK-Egr-1 pathway.[32] Remarkably, toll-like receptors (TLRs) related to the innate immune response have also been concerned in ZnO NP-induced expression of the proinflammatory cytokines such as TNF-α.[33] Furthermore, inflammatory reactions causing upregulation of IL-1 β, IL-6, and TNF-α strongly depend on mitogen-activated protein kinase (MAPK) signaling mediated by TLR6.[34]

CONCLUSION

It was concluded that the ZnO NPs administration caused toxic effects on the histological structure of the male albino rat lung. These effects were route and dose-dependent, being more obvious after intranasal administration.

Recommendation

It is recommended to limit the dose of ZnO NPs to the level that just meets the need of their usage in the medical field. Further studies on the outcome of ZnO NPs on other organs are needed. Finally, it is recommended to do further evaluation of the toxicity of ZnO NPs using diverse sizes, duration, and possible reversible effects after their cessation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.