Background: Nanoparticles of zinc oxide (ZnO-NPs) are frequently implemented in cosmetics, additives, and electronic devices. Moreover, their applications extend to water treatment, drug delivery, and cancer therapy. As a result, NP toxicity became an essential subject in biosafety research. Aim: Using histological and immunohistochemical analysis, we attempted to investigate whether ascorbate (”vitamin C”) (VC) could protect liver, lung, and spleen tissues from ZnO-NP systemic toxicity. Materials and Methods: Rats were classified as control group, NP group injected intraperitoneally (IP), once by dissolved ZnO-NPs (200 mg/kg), and NP + VC group injected IP, once by dissolved ZnO-NPs (200 mg/kg) and then ingested 100 mg/kg of VC orally. Blood samples were collected. Liver, lung, and spleen specimens were prepared for light, electron microscopic, and immunohistochemical analysis. Results: In comparison to the control group, the NP group's liver enzyme, i.e. aspartate transaminase and alanine transaminase, values and counts of white blood cells (WBCs) were higher on the 7th day, but their red blood corpuscle (RBC) count, hemoglobin (Hgb) level, platelet count, and albumin values were lower. Histopathological analysis of liver, lung, and spleen tissues showed severe toxicity manifested by cell apoptosis, mononuclear cell infiltration, dilated blood vessels, and hemorrhage. In addition, the NP group showed a significantly higher expression of Ki67 and caspase-3 immunoreactivity. The biochemical, hematological, and histopathological results of the NP + VC group improved overall, reflecting VC's protective effect against systemic toxicity. Conclusion: Our study revealed that ascorbate (VC) inhibited the systemic toxicity prompted by ZnO-NPs in lung, liver, and spleen tissues, indicating its importance for future treatment with ZnO-NPs.
Keywords: Liver, lung, nanoparticles of zinc oxide, spleen, Vitamin C
|How to cite this URL:|
Osman A, Afify SM, Frag A, Alghandour SM. Histological assessment of systemic toxicity induced by zinc oxide nanoparticles and the prophylactic potency of ascorbate in albino rats. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Feb 8]. Available from: https://www.jmau.org/preprintarticle.asp?id=362510
| Introduction|| |
The field of nanotechnology constitutes a modern sector of science that focuses on handling NPs with sizes between 1 to 100 nanometers. Since NPs are implemented in many fields, including pharmaceuticals, electronics, biomedicine, and cosmetics, their use has spread immensely. Meanwhile, their toxic and damaging side effects continue to increase. In consequence, the exposure of humans to NPs and their harmful effects became unavoidable.
Nanoparticles of zinc oxide (ZnO-NPs) are among the prevailing metal NPs with antimicrobial properties. In this sense, they have been utilized to keep food fresh longer and prevent its deterioration. Sunscreens are also prepared using ZnO-NPs due to their powerful ability to block ultraviolet radiation. Accordingly, ZnO-NPs commercially exist on a wide scale as a constituent of food additives, food packaging, and cosmetics. Moreover, their applications extend to water treatment, drug delivery, and cancer therapy. In this context, biosafety research regarding nanoparticle toxicity has become increasingly important.
Upon entering the bloodstream, ZnO-NPs settle in organs as zinc ions. Zinc ions are built up mainly in the liver, but they can also spread to other organs such as the lungs and spleen.
It is believed that the main factor causing cytotoxicity is the high amount of zinc ions. Chemical composition and morphology determine the toxicity of NPs, as well as their physicochemical characteristics.
Vitamin C (VC), also known as ascorbic acid-ascorbate, is an essential micronutrient for humans. VC is a powerful antioxidant that neutralizes oxidative stress, making it an essential substance. Recently, research has been shining the light on the benefits of antioxidants and their capacity of prevention and alleviation of damaging effects caused by the NPs.
This study investigates the in vivo histological systemic toxicity of ZnO-NPs and the potential preventive effects of VC on adult male albino rats' livers, lungs, and spleens.
| Materials and Methods|| |
Drugs and dosage
Intraperitoneally (IP) administration of ZnO-NPs was performed using distilled water (DW) given as a 200 mg/kg dose once.
Characterization of the particles
Chemical techniques (hydrothermal) were used to create ZnO-NPs from original precursors. The original precursors were obtained from Sigma-Aldrich Corporation (St. Louis, USA). Through transmission electron microscopy (TEM) (Tokyo, Japan), the prepared particles were analyzed.
VC “ascorbate” was provided by Kahira Pharmaceuticals and Chemicals, Egypt, in a commercial product called C-Retard 500 mg in the form of capsules. The content of the capsules was added to DW. Animals were given 100 mg/kg of the drug by oral ingestion once.,
Thirty-two male albino rats were kept at Kafrelsheikh University Animal House. Separately, rats weighing about 180–205 g (12 weeks old) were placed in metal cages with wire-grid floors at 25°C and 12-h cycles of light and darkness. Free water was available with a regular laboratory diet. The animals were housed according to directions of the Animal Ethical Committee of the Faculty of Medicine, Kafrelsheikh University. Animal Ethical Committee of the Faculty of Medicine, Kafrelsheikh (Approval code MKSU 50- 9- 7, Approval date 3- 9-2021).
Three groups of rats were assembled at random as follows:
- Control group: 12 rats were given DW as a vehicle in the same dosage and delivery method (IP/and oral) as the corresponding experimental groups
- NP group: 10 rats received IP injection, once by 1 ml of dissolved 200 mg/kg ZnO-NPs in DW
- NP + VC group: 10 rats received IP injection, once by 1 ml of dissolved 200 mg/kg ZnO-NPs in DW simultaneously with 100 mg/kg of VC dissolved in 1ml DW, orally ingested via gastric gavage.
All the animals were sacrificed after 7 days.
At the end of the experimentation, capillary tubes were used to collect samples from retro-orbital blood to measure the following parameters:
- Biochemical parameters: Albumin, aspartate transaminase (AST), and alanine transaminase (ALT) enzyme serum levels
- Hematological parameters: Hgb level, RBC count, platelet (PLT) count, white blood cell (WBC) count, and percentage of neutrophils, eosinophils, basophils, lymphocytes, and monocytes
- Tests were conducted at Kasr Al-Ainy Medical Hospital Clinical Pathology Department.
Xylazine (10 mg/kg) was used in a combination with ketamine (80 mg/kg) for anesthesia of the animals. The liver, lungs, and spleen were quickly removed and fixed immediately for the following studies:
The dissected organs were preserved for around 24 h in a 10% buffered formalin, dehydrated, cleared, and paraffin-impregnated for blocking. Hematoxylin and eosin (H and E) was used to stain serial slides of 5 μm thickness.
Sections of the specimens were immunostained using the following primary antibodies:
- Rabbit anti-Ki67 polyclonal antibody (1:1000, #ab66155, Abcam) for detection of proliferating cell population
- Rabbit anti-caspase-3 monoclonal antibody (1:200, #ab184787, Abcam) for detection of apoptotic cells.
The immunoperoxidase technique was then applied using the secondary antibody (Vectastain Elite ABC HRP Kit Peroxidase Universal, Burlingame, CA).
Electron microscopic studies
For TEM, small fragments from the liver, lung, and spleen were kept in buffered glutaraldehyde (2.5%), dehydrated, and embedded in resin. Toluidine blue staining was applied to semithin sections (1 μm) before being utilized to select the fields for TEM examination. Uranyl acetate and lead citrate were used to stain ultrathin slices (60–80 nm) for examination by (JEOL JEM-2100, Jeol Ltd., Tokyo, Japan) in the electron microscopy unit at the Faculty of Science, Alexandria University, Egypt, and by TEM JEOL (JEM-1400 TEM) and CCD camera, model AMT, at Electron Microscope Research Unit, Faculty of Agriculture, Cairo University.
Histomorphometric measurements were performed using Image Analyzer Leica Qwin 500 (LEICA Imaging Systems Ltd., London). The measurements included:
The mean area percentage of Ki67-positive cells in liver, lung, and spleen. The mean area percentage of caspase-3-positive cells in liver, lung, and spleen.
The means and standard deviation (SD) were applied to express the morphometric data. The statistical analysis was conducted using version 23 of SPSS software (SPSS, Chicago, IL, USA). For comparison between the groups, one-way analysis of variance was used, followed by a post hoc Tukey test. The findings are statistically significant when the P value is less than 0.05.
| Results|| |
One mortality was reported throughout the experiment from the NP group. Results of all rats of the control group were comparable to each other.
- Characterization of the particles: ZnO-NPs that were examined using TEM. ZnO-NPs exhibited a wide range of shapes, with the most common being spherical
- TEM was also used to determine each nanoparticle's size distribution [Figure 1]a and [Figure 1]b.
|Figure 1: (a) An electron micrograph showing the shape and size of ZnO-NPs by TEM (TEM, ×60.000). (b) Size distribution of ZnO-NPs determined by TEM. ZnO-NPs: Nanoparticles of zinc oxide, TEM: Transmission electron microscopy|
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The NP group showed a significantly lower serum level of albumin when compared to the control group, while the NP + VC group and the control group did not differ significantly regarding this parameter. Nonetheless, the NP + VC group demonstrated a significantly higher level of albumin in comparison with the NP group. A higher significant level of AST and ALT enzymes was observed in the NP group and NP + VC group than in the control group. However, the NP + VC group showed lower significant measures compared to the NP group [Figure 2]a.
|Figure 2: Mean values of biochemical and hematological parameters in control and experimental groups. (a) Mean values of albumin, AST, and ALT serum levels. (b) Mean values of Hgb level, RBCs, PLTs, and WBC count. (c) Mean values of neutrophils, eosinophils, basophils, lymphocytes, and monocytes%. AST: Aspartate transaminase, ALT: Alanine transaminase, Hgb: Hemoglobin, RBCs: Red blood corpuscles, PLT: Platelet, WBCs: White blood cells|
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The NP group showed significantly lower values of Hgb level, RBC count, and PLT count when compared to the control group. These parameters displayed no significant difference between the NP + VC group and the control group; however, the NP + VC group demonstrated significantly higher values in comparison with the NP group. Contrarily, the WBC count showed the opposite pattern of changes. As compared with the control group, the NP and NP + VC groups displayed significantly higher values, while the NP + VC group displayed significantly lower values [Figure 2]b.
Both the NP group and the NP + VC group had significantly higher percentages of neutrophils, lymphocytes, and monocytes in comparison to the control group. Nonetheless, neutrophil and lymphocyte percentages significantly reduced in the NP + VC group compared to the NP group while the percentage of monocytes recorded a significant increase. The percentages of eosinophils and basophils in the three groups did not differ significantly [Figure 2]c.
Hematoxylin and eosin staining
Typical lobule and hepatocyte structures were observed in liver sections from the control group. The hepatic lobule comprised a central vein, radially organized hepatocytes in linear cords with hepatic blood sinusoids in between, in addition to the presence of portal tracts. Normal hepatocytes exhibited central rounded vesicular nuclei with prominent nucleoli [Figure 3]a and [Figure 3]b.
|Figure 3: Photomicrograph of H- and E-stained liver sections. (a and b) Control group showing typical architectural pattern of hepatic lobule demonstrating hepatocytes (yellow arrows) exhibiting central rounded vesicular nuclei with prominent nucleoli and organized in cords radiating around a central vein (asterisks). Hepatic blood sinusoids (blue arrows) are radially arranged in between the cords. A PT is noticed. (c and d) NP group showing marked destruction of the hepatic architectural pattern demonstrating disorganization of the hepatic cords (curved arrows), thick CF, hemorrhage, markedly congested and dilated blood vessels (c), and some mononuclear inflammatory cell aggregations (green arrows). Groups of hepatocytes with deeply acidophilic cytoplasm (circles), hepatocytes with marked cytoplasmic vacuolation, and pyknotic nuclei (black arrows) are illustrated. Dilated blood sinusoids in some areas (blue arrows) and brown deposits inside von Kupffer cells (red arrows) are noticed. (e and f) NP + VC group showing some areas with normal structural pattern of hepatic lobule and normal appearance of hepatocytes (yellow arrows) while other areas demonstrate disorganization of the hepatic cords (curved arrow). Some hepatocytes exhibit marked cytoplasmic vacuolation with pyknotic nuclei (black arrow) and few ones have deeply acidophilic cytoplasm (circles). A PT demonstrates an enlarged and dilated portal vein (v). A slightly dilated central vein (asterisk) contains few mononuclear inflammatory cells (green arrows) which are also noticed near the PT. Slightly dilated hepatic sinusoids (blue arrow) and brown pigments in von Kupffer cells (red arrows) are observed. (H and E: a, c and e, ×100; b, d and f, ×200). PT: Portal tract, CF: Collagen fiber septa, H: Hemorrhage, NPs: Nanoparticles, H and E: Hematoxylin and eosin|
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The NP group sections showed great destruction of hepatic architectural organization illustrating loss of the hepatic cord arrangement, groups of hepatocytes with deeply acidophilic cytoplasm and others with marked cytoplasmic vacuolation and pyknotic nuclei, aggregations of some mononuclear inflammatory cells, collagen fiber deposition, sinusoidal dilatation, areas of hemorrhage, and markedly dilated congested blood vessels. Brown deposits in von Kupffer cells were noticed in the lining of some blood sinusoids [Figure 3]c and [Figure 3]d.
Examination of the NP + VC group revealed some areas with a normal architectural organization of hepatic lobules and normal histological structure of hepatocytes. However, some areas showed a disordered arrangement of the hepatic cord. Some hepatocytes exhibited marked vacuolation of cytoplasm and pyknotic nuclei while few ones had deeply acidophilic cytoplasm. Some portal tracts displayed dilatation of their veins. Few central veins showed slight dilatation with few mononuclear inflammatory cells in their lumina as well as near some portal tracts. Slight dilatation of some blood sinusoids alongside the presence of brown pigments in their lining von Kupffer cells was also seen [Figure 3]e and [Figure 3]f.
A typical architectural pattern was observed in lung sections from the control group, which exhibited extremely thin type I pneumocytes and larger type II pneumocytes, alveolar sacs made up of groups of grouped alveoli, and interalveolar septa between adjacent alveolar spaces, which are rich in blood capillaries, and air-conducting ducts including the terminal bronchioles [Figure 4]a and [Figure 4]b.
|Figure 4: Photomicrograph of H- and E-stained lung sections. (a and b) The control group shows a typical alveolar architecture (yellow arrows), equipped with thin and large pneumocytes (bifid arrows), alveolar sacs (s), interalveolar septa (wavy arrow) comprising BCs, in addition to normal histological structure of the terminal bronchiole (asterisk). (c and d) NP group illustrating a dilated terminal bronchiole (asterisk) with shed epithelial cells in its lumen (curved arrow). Mononuclear inflammatory cell infiltration (green arrows) is seen around the terminal bronchiole (asterisk), inside thickened interalveolar septa (right angle arrows) present between the alveoli (yellow arrows) and in the lumen of congested and dilated blood vessel (c) and BCs. (e and f) NP + VC group demonstrating normal histological structure of the terminal bronchiole (asterisk), alveoli (yellow arrows), alveolar sacs (s), and interalveolar septa (wavy arrows). Few congested blood vessels (c) are observed. Inflammatory cellular aggregates (green arrows) are noticed near the terminal bronchiole (asterisk), inside slightly thickened interalveolar septa (right angle arrows) and in few BCs (H and E: a, c and e, ×100; b, d and f, ×200). BC: Blood capillary, NPs: Nanoparticles, H and E: Hematoxylin and eosin|
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The NP group sections demonstrated dilatation of the terminal bronchioles with shed epithelial cells in their lumina. Mononuclear inflammatory cell infiltration was evident in peribronchial regions, thickened interalveolar septa, and lumina of congested and dilated blood vessels and capillaries [Figure 4]c and [Figure 4]d.
Examination of the NP + VC group revealed the normal histological structure of the terminal bronchioles, alveoli, alveolar sacs, and interalveolar septa in most areas of the lung sections; however, few areas demonstrated congested blood vessels, alongside inflammatory cellular aggregates in few peribronchial regions, inside slightly thickened interalveolar septa and in few blood capillaries [Figure 4]e and [Figure 4]f.
Examination of spleen sections of the control group revealed typical structure of white pulp and red pulp with trabeculae in between. The white pulp consisted of lymphatic tissue. It comprised a germinal center, a marginal zone, and a central arteriole. The red pulp comprised splenic sinuses between its splenic cords [Figure 5]a and [Figure 5]b.
|Figure 5: Photomicrograph of H- and E-stained spleen sections. (a and b) WP and RP typical composition of the control group. Both types of pulp contain trabeculae (t). The boxed area in a is depicted with a higher magnification in b illustrating the arrangement of lymphocytes (yellow arrow) in WP forming a GC and a MZ. A CA is noticed. The RP comprises SS. (c and d) NP group demonstrating loss of splenic histological organization with a slightly deformed WP showing some distortion in the arrangement of lymphocytes (yellow arrow). Clusters of mononuclear inflammatory cells (green arrows) are seen infiltrating the RP as well as dilated SS. Brown deposits inside macrophages (red arrows) are detected in RP. (e and f) NP + VC group showing almost normal structure of WP. Aggregates of mononuclear inflammatory cells (green arrows) are noticed in RP. Slightly dilated SS and brown deposits in macrophages (red arrows) are observed (H and E: a, c and e, ×100; b, d and f, ×200). WP: White pulp, RP: Red pulp, GC: Germinal center, MZ: Marginal zone, CA: central arteriole, SS: Splenic sinuses, NPs: Nanoparticles, H and E: Hematoxylin and eosin|
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The NP group sections revealed a loss of splenic architectural pattern. Some deformed white pulps showed some distortion of lymphatic architecture, mononuclear inflammatory cell aggregations in red pulp infiltrating the splenic cords as well as dilated splenic sinuses. Brown deposits inside macrophages in red pulp were observed [Figure 5]c and [Figure 5]d.
Sections of the NP + VC group showed an almost normal histological appearance of white pulp. However, some areas in the red pulp showed clusters of mononuclear inflammatory cells and slightly dilated splenic sinuses in addition to macrophages containing brown deposits [Figure 5]e and [Figure 5]f.
Immunohistochemical stained sections
The control group of immunostained liver sections with anti-Ki67 antibody showed weak nuclear immunoreactivity in few hepatocytes and endothelial cells. Strong nuclear immunoreaction was portrayed in the NP group in numerous hepatocytes as well as few endothelial cells lining blood sinusoids. The NP + VC group showed moderate nuclear immunoreactivity in some hepatocytes and few endothelial cells [Figure 6]a, [Figure 6]b, [Figure 6]c. Lung sections of the control group revealed negative immunoreactivity while strong nuclear immunoreaction was portrayed in the NP group in numerous cells of inflammatory infiltrates in addition to some cells of bronchiolar epithelium. The NP + VC group showed moderate nuclear immunoreactivity in some inflammatory cellular aggregates and few cells in bronchiolar and alveolar epithelium [Figure 6]d, [Figure 6]e, [Figure 6]f. The control group of spleen sections demonstrated mild nuclear immunoexpression in some cells of red and white pulp, mainly in the germinal center and less in the marginal zone. The NP group revealed strong nuclear immunoreaction in numerous cells scattered throughout deformed white pulps, especially in the marginal zone, alongside their wide dispersal in the red pulp. The NP + VC group showed moderate nuclear immunoreactivity in some cells, mainly in almost normally organized germinal centers in white pulps, less in marginal zone, in addition to few cellular clusters in red pulps [Figure 6]g, [Figure 6]h, [Figure 6]i.
|Figure 6: Photomicrograph of immunostained sections for Ki67 of the liver, lung, and spleen: (a-c) liver sections, (a) Control group showing weak nuclear immunoreactivity in few hepatocytes (yellow arrows) and endothelial cells (blue arrow) near the PT. (b) NP group portraying strong nuclear immunoreaction in numerous hepatocytes (yellow arrows) as well as endothelial cells (blue arrow) lining blood sinusoids near the PT. (c) NP + VC group showing moderate nuclear immunoreactivity in some hepatocytes (yellow arrows) and endothelial cells (blue arrow) close to the PT. (d-f) Lung sections, (d) Control group demonstrating negative immunoreactivity. (e) NP group revealing strong nuclear immunoreaction in numerous cells of inflammatory infiltrates (green arrows) in addition to some epithelial cells (curved arrows) lining the terminal bronchiole (asterisk). (f) NP + VC group showing moderate nuclear immunoreactivity in some inflammatory cellular aggregates (green arrows) and few epithelial cells (curved arrow) lining the terminal bronchiole (asterisk) as well as alveolar epithelium (bifid arrow). (g-i) Spleen sections, g: Control group demonstrating mild nuclear immunoexpression in some cells (black arrows) of RP and WP, mainly in the GC and less in the MZ. h: NP group revealing strong nuclear immunoreaction in numerous cells (black arrows) scattered throughout deformed WP, especially in the MZ, alongside their wide dispersal in the RP. i: NP + VC group showing moderate nuclear immunoreactivity in some cells (black arrows) mainly in almost normally organized GC in WP, less in MZ, in addition to few cellular clusters in RP (anti-Ki67 immunostaining, ×200). PT: Portal tract, NPs: Nanoparticles, RP: Red pulp, WP: White pulp, GC: Germinal center, MZ: Marginal zone|
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The control group sections of liver immunostained with anti-caspase-3 antibody revealed weak cytoplasmic immunoreaction in scanty hepatocytes, while the NP group sections revealed strong cytoplasmic immunoexpression in numerous hepatocytes, few endothelial cells lining blood sinusoids as well as some mononuclear inflammatory cellular infiltrates. The NP + VC group tissues displayed moderate cytoplasmic immunoexpression in some hepatocytes and few endothelial cells lining the blood sinusoids [Figure 7]a, [Figure 7]b, [Figure 7]c. Lung control sections displayed negative immunoreactivity. The NP group sections revealed strong cytoplasmic immunoexpression in numerous inflammatory cellular aggregations in addition to few cells of bronchiolar and alveolar epithelium. The NP + VC group tissues displayed moderate cytoplasmic immunoreactivity in some inflammatory cellular infiltrates and few cells of alveolar epithelium [Figure 7]d, [Figure 7]e, [Figure 7]f. The control group sections of spleen sections showed faint cytoplasmic immunoexpression in scarce cells in white and red pulps. The NP group sections revealed strong cytoplasmic immunoexpression in numerous cells scattered throughout deformed white pulps and red pulps in spleen sections. The NP + VC group tissues displayed moderate cytoplasmic immunoreaction in some cells in white and red pulps [Figure 7]g, [Figure 7]h, [Figure 7]i.
|Figure 7: Photomicrograph of immunostained sections for caspase-3 of the liver, lung, and spleen: (a-c) liver sections, (a) Control group: revealing weak cytoplasmic immunoreaction in one hepatocyte (yellow arrow) near the central vein (asterisk). (b) NP group illustrating strong cytoplasmic immunoexpression in numerous hepatocytes (yellow arrows), endothelial cells (blue arrow) lining blood sinusoids as well as some mononuclear inflammatory cellular infiltrates (green arrow) near the PT. (c) NP + VC group displaying moderate cytoplasmic immunoexpression in some hepatocytes (yellow arrows) and few endothelial cells (blue arrows) lining the blood sinusoids near the PT. (d-f) lung sections, (d) Control group: displaying negative immunoreactivity. € NP group revealing strong cytoplasmic immunoexpression in numerous inflammatory cellular aggregations (green arrows) in addition to few epithelial cells (curved arrow) lining the terminal bronchiole (asterisk) as well as alveolar epithelium (thin arrows). (f) NP + VC group showing moderate cytoplasmic immunoreactivity in some inflammatory cellular infiltrates (green arrows) and few cells of alveolar epithelium (thin arrows). (g-i) Spleen sections, g: Control group showing faint cytoplasmic immunoexpression in scarce cells (black arrows) in WP and RP. h: NP group revealing strong cytoplasmic immunoexpression in numerous cells (black arrows) scattered throughout deformed WP and RP. (i) NP + VC group displaying moderate cytoplasmic immunoreaction in some cells (black arrows) in WP and RP (anti-caspase-3 immunostaining, ×200). NPs: Nanoparticles, PT: Portal tract, WP: White pulp, RP: Red pulp|
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Electron microscopic sections
Ultrathin sections of the control group's liver tissue showed the hepatocytes' typical histological structure containing euchromatic nucleus with prominent nucleolus and an intact and distinct nuclear envelope. The cytoplasm contained numerous mitochondria with spherical to ovoid shapes, rough endoplasmic reticulum (rER) with well-developed, regular, and closely packed flattened cisternae studded with ribosomes, lysosomes, and numerous electron-dense glycogen granules. Bile canaliculi were seen between the cell surfaces of adjacent hepatocytes [Figure 8]a.
|Figure 8: Electron micrograph of ultrathin sections of liver showing: (a) Typical histological structure of a hepatocyte from the control group with an intact and distinct NE and an euchromatic nucleus (n) with prominent nucleolus (n). The cytoplasm contains numerous mitochondria (m) with spherical to ovoid shapes, rER with well-developed, regular, and closely packed flattened cisternae studded with ribosomes, LY, and numerous electron-dense glycogen granules (g). Note the presence of a bile canaliculus (blue triangle) between adjacent hepatocytes [x5000]. b: NP group demonstrates marked degenerative changes which appear in the form of electron-lucent areas of cytoplasmic vacuolations (v), deformed mitochondria (m) with destructed cristae (blue bifid arrow), and vacuolations (blue-curved arrow), fragmentation (blue arrowhead) of rER. The nucleus (n) shows irregular and disrupted (blue wavy arrow) NE. Peroxisomes (p), and LY of various sizes and shapes are observed [x5000]. c: Cytoplasm of a hepatocyte from NP group shows marked degenerative changes in the form of electron-lucent wide areas of cytoplasmic vacuolations (v), deformed mitochondria (m) with destructed cristae (blue bifid arrow) and vacuolations (blue curved arrow), and fragmentation (blue arrowhead) of rER. Note the presence of peroxisomes (p), and various sizes and shapes of LY (×6000). (d) A hepatocyte from NP + VC group shows some histological changes in the form of cytoplasmic vacuolations (v) which appear as cytoplasmic electron-lucent areas containing a material of medium electron density, fragmentation (blue arrowhead) of rER in addition to clumped and condensed heterochromatin (blue arrow) in the nucleus (n). The presence of mitochondria (m), LY, as well as LDs is noted (×4000). (e) A hepatocyte from NP + VC group showing a mostly normal histological structure exhibiting an euchromatic nucleus (n) and intact NE. Mitochondria (m) with normal appearance and rER with closely packed flattened cisternae studded with ribosomes are seen in the cytoplasm. Few rER cisternae show fragmentation (blue arrowhead). Areas of cytoplasmic vacuolation (v) are also seen (TEM X, ×8000). NE: Nuclear envelope, rER: Rough endoplasmic reticulum, LY: Lysosomes, NPs: Nanoparticles, NE: Nuclear envelope, LD: Lipid droplet, NE: Nuclear envelope|
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The NP group sections demonstrated marked degenerative changes appeared in the form of electron-lucent areas of cytoplasmic vacuolations, deformed mitochondria with destructed cristae and vacuolations, fragmented rER, and irregular and disrupted nuclear envelope. Peroxisomes and lysosomes were observed [Figure 8]b and [Figure 8]c.
Liver sections from the NP + VC group showed less severe changes in some hepatocytes comprising cytoplasmic vacuolations which appeared as cytoplasmic electron-lucent areas containing a material of medium electron density, fragmentation of rER, in addition to clumped and condensed heterochromatin in some nuclei. The presence of lysosomes and mitochondria was noted, as well as lipid droplets [Figure 8]d. However, most of hepatocytes exhibited nearly normal histological structure characterized by nucleus with euchromatin and distinct nuclear membrane. Mitochondria with normal appearance and rER with closely packed flattened cisternae studded with ribosomes were noticed although few rER cisternae showed some fragmentations. Few areas of cytoplasmic vacuolations were also seen [Figure 8]e.
Normal architecture of lung sections was demonstrated in the control group. Type I thin pneumocytes line the alveolar wall, while type II large pneumocytes line the alveolar wall. The air–blood barrier consisted of the cytoplasm of endothelial cells lining blood capillaries, the cytoplasm of pneumocytes type I, and the fused basal laminae of both types of these cells. Interstitial macrophages were noticed containing cytoplasmic vacuoles and pseudopodia. Pneumocytes type II with numerous lamellar bodies in the cytoplasm, euchromatic nuclei, rER, and few short microvilli were observed [Figure 9]a and [Figure 9]b.
|Figure 9: Electron micrograph of ultrathin sections of lung showing: (a) Normal architecture of the lung tissue of control group is illustrated. Two types of cells are seen lining the wall of the alveoli (a), the thin P1 and the large P2, which contain numerous LBs in their cytoplasm. The air–blood barrier is seen formed of the cytoplasm of an EC lining a BC containing RBCs, cytoplasm of P1, and their fused basal laminae (yellow right angel arrow). An interstitial Mac is noticed containing cytoplasmic vacuoles (v) and Ps (×2000). (b) A P2 from control group showing numerous LBs in its cytoplasm with their characteristic lamellar pattern (yellow double arrows), euchromatic nucleus (n), rER, and a free surface with few short Mv (×12000). (c) A section in the lung from NP group showing an air–blood barrier formed of cytoplasm of an EC lining a BC congested with RBCs, thick fused basal laminae (yellow right angel arrow), and swollen and enlarged P1. Note the presence of a fibroblast (f) and CFs in thickened IS present between the alveoli (a) (×2000). (d) P2 in the lung section from NP group showing irregular outline, irregular heterochromatic nucleus (n), diminished Mv, loss of the pattern of LBs, and few cytoplasmic vacuoles (v). Thick IS is observed containing deposited CFs and a fibroblast (f) with branches (bifid yellow arrow), euchromatic nucleus (n), and prominent nucleolus (n) (×2000). (e) Thick area of an IS in lung section from NP group showing wide large areas of CF deposition, a group of fibroblasts (f) and a BC congested with RBCs (×3000). (f) A section from NP + VC group revealing a few alterations in the structure of a P2 showing vacuolated and empty LBs, irregular and indented (yellow arrow) nucleus (n), few small vacuolations (v) in the cytoplasm, and slightly diminished surface Mv. A P1 shows normal flattened appearance and few cytoplasmic vacuolations (v) (×2000). (g) IS in a lung section from NP + VC group showing BC slightly congested with RBCs, and few deposited CFs. Two fibroblasts (f) are noticed An alveolar Mac is seen in the lumen of an alveolus (a). It is characterized by a large heterochromatic nucleus (n) with indentation (yellow arrow), Ps, vacuoles (v), and numerous Ly of various shapes and sizes (TEM X, ×2000). P1: Pneumocyte type I, P2: Pneumocyte type II, LB: Lamellar body, EC: Endothelial cell, BC: Blood capillary, RBCs: Red blood corpuscles, Mac: Macrophage, Ps: Pseudopodia, rER: Rough endoplasmic reticulum, Mv: Microvilli, CF: Collagen fibers, IS: Interalveolar septum|
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Lung sections of the NP group showed some histological changes in lung tissue structure such as air–blood barriers which were formed of the cytoplasm of endothelial cells lining congested blood capillaries, thick fused basal laminae, and enlarged pneumocyte type I. Fibroblasts were noticed in thickened interalveolar septa. They were branched and surrounded by deposited collagen fibers [Figure 9]c. Pneumocytes type II showed irregular outline, irregular heterochromatic nuclei with diminished microvilli, few cytoplasmic vacuoles, and lamellar bodies with disrupted architecture. Collagen deposition and interstitial fibroblasts were detected in the interalveolar septa. They were branched cells that exhibited euchromatic nuclei with prominent nucleoli and were surrounded by collagen secreted by them [Figure 9]d. Thick areas of interalveolar septa were encountered showing wide large areas of collagen deposition around groups of fibroblasts in addition to congested blood capillaries [Figure 9]e.
Sections from the NP + VC group revealed less alterations in the histological structure. Some pneumocytes type II showed changes in the structure of their lamellar bodies; some appeared vacuolated and others were empty. Some nuclei were indented. Surface microvilli were slightly diminished and the cytoplasm showed few small vacuolations. Most pneumocytes type I had an almost normal flattened appearance; however, the cytoplasm showed few small vacuolations [Figure 9]f. Some interalveolar septa contained slightly congested blood capillaries, few small areas of collagen fiber deposition, and fibroblasts. Alveolar macrophages were occasionally encountered in the lumen of alveoli. They demonstrated large heterochromatic and indented nuclei, pseudopodia, vacuoles, and numerous lysosomes [Figure 9]g.
Ultrastructurally, spleen sections from the control group revealed closely packed small lymphocytes with heterochromatic nuclei in the white pulp. Reticular fibers were also seen [Figure 10]a. The red pulp contained small lymphocytes with heterochromatic nuclei, and plasma cells which exhibited large nuclei with clumps of heterochromatin alternating with euchromatin, in addition to well-developed rER. Splenic sinuses filled with RBCs were demonstrated [Figure 10]b.
|Figure 10: Electron micrograph of ultrathin sections of spleen showing: (a) A spleen section in white pulp from control group revealing closely packed small lymphocytes (L) with heterochromatic nuclei (N). RFs are also noticed [x8000]. (b) Red pulp of a spleen section from control group revealing a small lymphocyte (L) with heterochromatic nucleus (N) and a large, ovoid PC with large nucleus (N) showing clumps of heterochromatin (blue arrow) alternating with euchromatin (blue asterisk), in addition to an extensive, well-developed rER. SS filled with RBCs are demonstrated (×10000). (c) A section of spleen from NP group showing congested SS filled with RBCs. A Mac with extended Ps, irregular nucleus (N), cytoplasmic vacuoles (V), and an engulfed RBC is illustrated. The Mac is seen phagocytosing (green bifid arrows) electron-dense particles (green-circled area). A Mon with large euchromatic nucleus (N) with its characteristic indentation (green arrow) as well as cytoplasmic vacuolations (V) is demonstrated. (d) Spleen section from NP group showing parts of two adjacent Mac, one demonstrates a large Ph containing electron-dense particles in addition to multiple lysosomes (L), the other shows an irregular heterochromatic nucleus (N), rER with disrupted organization (green arrowhead), and cytoplasmic vacuoles (V). Note the presence of RFs between the two cells (×5000). (e) A section in white pulp of spleen tissue from NP + VC group showing small lymphocytes (L) with variable grades of chromatin condensation in their nuclei (N), large aL with euchromatic nuclei (N), RCs, a Mac, PC, and a MC filled with its characteristic intensely stained granules (green arrows). Note the presence of RFs among the cells (×800). (f) Higher magnification of the green boxed area in micrograph e showing a Mac exhibiting an irregular outline with Ps, irregular nucleus (N), and multiple cytoplasmic vacuoles (V) as well as a Ph containing electron-dense particles (green-circled area). A RC is demonstrated containing a hyperchromatic nucleus (N) and cytoplasmic processes (green wavy arrows). A part of a small lymphocyte (L) is noticed with a heterochromatic nucleus (N) as well as a part of a PC showing a heterochromatic nucleus (N) and slight dilatations and disrupted appearance (green arrowhead) in its rER cisternae. Note the presence of RFs among the cells (TEM X, ×3000). RF: Reticular fiber, PC: plasma cell, SS: Splenic sinuses, RBCs: Red blood corpuscles, NPs: Nanoparticles, Mac: Macrophage, Ps: Pseudopodia, Mon: Monocytes, Ph: Phagosome, rER: Rough endoplasmic reticulum, aL: Activated lymphocytes, RC: Reticular cell, MC: Mast cell|
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Sections of spleen tissue from the NP group showed congested splenic sinusoids filled with RBCs. Degenerated lymphocytes with irregular outline and condensed chromatin in their pyknotic nuclei were noticed. Macrophages with extended pseudopodia, irregular nuclei, disrupted rER organization, multiple lysosomes, cytoplasmic vacuoles, and engulfed RBCs were also seen. Macrophages phagocytosing electron-dense particles were encountered and others with phagosomes containing electron-dense particles were also noticed. Monocytes with large euchromatic nuclei showing their characteristic indentation in the centrosphere region of the cell as well as cytoplasmic vacuolations were detected [Figure 10]c and [Figure 10]d.
Group NP + VC sections of the spleen showed small lymphocytes with variable grades of chromatin condensation and large activated lymphocytes with euchromatic nuclei, macrophages, reticular cells, plasma cells, and occasional mast cells in a background of reticular fiber framework. Macrophages demonstrated an irregular outline with pseudopodia, irregular nuclei, and multiple cytoplasmic vacuoles with occasional presence of phagosomes containing electron-dense particles. Reticular cells contained hyperchromatic nuclei and showed extended cytoplasmic processes. Sporadic plasma cells exhibited heterochromatic nuclei and slight dilatations in rER cisternae [Figure 10]e and [Figure 10]f.
Morphometric and statistical results
Mean values of area % of Ki67 [Figure 11]a and caspase-3 [Figure 11]b-immunopositive cells in liver, lung, and spleen recorded a significant increase in the NP group and NP + VC group as compared to the control group. In contrast to the NP group, a marked decrease was seen in the NP + VC group.
|Figure 11: Morphometric and statistical results in control and experimental groups in liver, lung, and spleen. (a) Ki67-immunopositive cells as a percentage of the mean area. (b) Mean area % of caspase-3-immunopositive cells|
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| Discussion|| |
The growing utilization of ZnO-NPs in wide array of applications has directed research to study their harmful impact on health. The spleen and liver were mentioned as being crucial target organs with the highest NP accumulation. According to reports, specialized vascularization and enhanced vascular permeability cause NPs to accumulate in the lungs. A nanoparticle's size has a significant effect on nanomaterials' capabilities. The nanoparticles' size influences their interactions with living cells, including their cytotoxicity. Because of their small size, nanoparticles can travel to different organs after being ingested. Hence, they can enter the bloodstream, brain, lung, kidney, spleen, liver, and gut after crossing the small intestine. Depending on the previous facts, we used ZnO-NPs with an average diameter of 29.4 nm to study the ZnO-NP cytotoxicity and the protective potential of VC in liver, lung, and spleen tissues.
According to the current study, ZnO-NPs disturb biochemical and hematological parameters, as well as alter the microscopic structure of liver, lung, and spleen tissues. A significant difference was found between the NP group and the control group with regard to serum albumin levels, whereas no significant differences were found in the NP + VC group.
On the other hand, the NP and NP + VC groups showed significantly higher values of AST and ALT enzymes. However, the NP + VC group recorded significantly lower levels in comparison to the rats that administered NPs alone. Researches recorded significant affection of liver function tests secondary to ZnO-NPs that are consistent with the results of the present study.,
One of the ways in which Zn-ONPs cause cytotoxic effects is through oxidative stress. In agreement with our outcomes, a recent research showed that VC prevented ZnO-NP-induced damage to the liver cells, and inhibited the rise in ALT and AST enzymes recorded in the ZnO-NP-intoxicated group.
Our present work detected significantly lower values of Hgb level, RBC count, and PLT count in the NP group when compared to the control group; however, these parameters recorded no significant change between the NP + VC group and the control group. That might be a direct result of harm to the liver and spleen, which play a big part in heme production, recycling old RBCs, and storage of PLT. Similarly, Yan et al., 2012, found that ZnO-NP-treated mice had lower Hgb levels and RBC indices, implying that the animals were anemic. ZnO-NPs were reported to lower the RBC count in rats which supports our findings. Furthermore, Abass et al., 2017, revealed that ZnO-NP utilization caused a decrease in count of RBCs and PLT. The capability of ZnO-NPs to cross cell membranes may clarify the RBC destruction.
On the other hand, WBC count in the current study showed significantly higher values in the NP and NP + VC groups when compared to the control group, and significantly lower values in the NP + VC group in comparison with the NP group. Neutrophils, lymphocytes, and monocytes percentages significantly increased in the NP and NP + VC groups compared to the control group. Neutrophil and lymphocyte percentages significantly decreased in the NP + VC group compared to the NP group while that of monocytes recorded a significant increase.
Due to the oxidative stress and lipid peroxidation-induced damage induced by these nanoparticles, an increase in WBC counts may reflect an inflammatory response. Oxidative stress is caused by acellular variables such as particle size, surface, and composition whereas cellular factors such as mitochondrial respiration, interface between NPs and the cell, and immune cell stimulation result in damage caused by reactive oxygen species (ROS). Researchers reported that VC administration partially inhibited ZnO-NP toxicity that results in an increase in WBC count. Contrarily, ZnO-NPs caused a significant decrease in WBCs including neutrophils. Although agreeing with our results, recent research recorded a significant rise in number of lymphocytes and monocytes. Interestingly, Fukui et al., 2015, attributed the preventive effect of ascorbic acid against inflammation triggered by ZnO-NPs, to suppression of the production of ROS as a result of stimulation of phagocytes as neutrophils.
Liver damage obtained by ZnO-NPs and reflected through the laboratory results was further confirmed by histopathological examination. Our results mirrored those of other previous studies.,,,
In our study, sections of liver stained with H and E from the NP group revealed the presence of aggregations of some mononuclear inflammatory cells. Hepatocytes with marked cytoplasmic vacuolation were detected in the current work as well as in ZnO-NP groups in other studies.,, In consistence with our findings, Hegazy et al., 2018, have previously demonstrated a marked increase in collagen fiber deposition in ZnO-NP-treated rats. This was attributed to the activation of fibroblasts as a consequence of oxidative stress.
In the current work, the NP + VC group revealed a certain degree of amelioration of hepatic tissue in some areas that exhibited almost normal histological structure. Agreeing with our work, histological analysis by Bayat et al., 2021, revealed that VC administration mitigated the liver damage caused by ZnO-NP-driven oxidative stress in rats. VC intake decreases the production of ROS which causes toxic damage. It also lowers the lipid peroxide's production promoted by oxidative stress.
Researchers discovered that ultrathin liver sections of the NP group showed significant deterioration in hepatocytes, including cytoplasmic vacuolations, mitochondria that were destroyed, fragmented rER, as well as irregular and disrupted nuclear envelopes. The presence of peroxisomes and lysosomes was noted. The NP + VC group showed less severe changes in some hepatocytes such as clumped and condensed heterochromatin, while most of hepatocytes exhibited nearly normal histological structure.
Ultrastructure findings of Abu-Dief et al., 2018, agreed with our previously depicted picture. Some degenerative changes in hepatocytes of titanium dioxide NP-treated rats were similar to ours, such as cytoplasmic vacuoles and manifestations of apoptosis in the form of the presence of clumps of heterochromatin and irregular nuclear membranes. Interestingly, the presence of increased peroxisomes was linked to increased hepatocyte proliferation along with playing a part in lipid metabolism and hydrogen peroxide detoxification.
Parallel to our work, ultrastructural results of Shamel et al., 2021, demonstrated almost normal mitochondria, rER, and nuclei in cells of submandibular glands in silver NP-intoxicated rats due to the antioxidant effects of VC through lowering of ROS. In addition, Julia Kaźmierczak-Barańska et al., 2020, have also explained that the capacity of VC to bind to and neutralize ROS ultimately leads to protecting from mitochondrial destruction, lipid oxidation, and DNA damage.
The histopathological changes in liver tissues were further confirmed by morphometric measurements of the mean area % values of Ki67 and caspase-3-immunopositive hepatocytes and endothelial cells. It showed a significant elevation in the NP group and NP + VC group in comparison to the control group. As compared to the NP group, the NP + VC group showed a significant decrease.
It had been established that the caspase cascades were stimulated by ZnO-NPs, resulting in apoptotic cell death through superoxide-triggered mitochondrial impairment. ZnO-NPs enhanced the immunoreactive area of the apoptotic protein bax as well as the expression of the hepatic caspase-3 gene. Antioxidant administration such as Vitamin E to ZnO-NP-treated rats has decreased the raised activity of caspase-3 in liver, which was attributed to its antiapoptotic properties.
It was observed in our H and E sections of the NP group lung tissue, dilated terminal bronchioles with shed epithelial cells and mononuclear inflammatory cell infiltrations in peribronchial regions, thickened interalveolar septa, and dilated capillaries. Most areas of the lung sections in the NP + VC group showed nearly normal histological structure, but few areas showed degenerative changes. An earlier study had demonstrated infiltration of macrophages and inflammatory cells around the terminal bronchioles in lung specimens exposed to ZnO-NPs. Only slight fibrosis was present after the inflammation. ZnO-NPs induce inflammatory responses and immune cell differentiation. Congested blood vessels, interstitial infiltration with inflammatory cells, and thickened interalveolar septa were all signs of lung injury caused by ZnO-NPs. Focal loss of epithelial integrity was reported in lung sections of the ZnO-NP-intoxicated group in many studies which were carried out on mice and rats.,
Thickening of interalveolar septa induced by ZnO-NPs was attributed to increased interstitial deposition of collagen fibers and obvious cellular infiltration with inflammatory cells. Congestion of blood vessels and cellular infiltration were explained by alteration of the vascular integrity leading to disturbance of the endothelial barrier and enhanced capillary permeability.
Ultrastructural examination of lung sections of the NP group in the present study showed some histological changes in lung tissue such as air–blood barriers with congested blood capillaries, thick fused basal laminae, and enlarged pneumocytes type I. Fibroblasts exhibiting euchromatic nuclei with prominent nucleoli and congested blood capillaries were noticed in thickened interalveolar septa as well as deposited collagen fibers. Pneumocytes type II showed irregular outline, irregular heterochromatic nuclei with diminished microvilli, few cytoplasmic vacuoles, and lamellar bodies with disrupted architecture. The NP + VC group revealed less alterations in the histological structure with partial improvement of the previously mentioned histopathology. Occasional alveolar macrophages with heterochromatic nuclei with indentation, vacuoles, and numerous lysosomes were encountered in the alveolar lumen.
Parallel to the previously portrayed picture, Elbakary et al., 2018, depicted similar alterations in lung tissue exposed to gold NPs. They showed pneumocytes type II with irregular, partially vacuolated, or empty lamellar bodies with lost surface microvilli. The vacuolations were attributed to increased sodium and water influx as a result of disruption in the membranes' function. Interalveolar septa contained congested blood capillaries and collagen fibers. Interstitial cells and inflammatory cells were also detected. These alterations were attributed by Elbakary et al., 2018, to impairment of the antioxidant defense mechanism, leading to the production of ROS. Fibroblast activation could explain the deposition of collagen fibers which agrees with the findings of previous work by Mohammed et al., 2022, on ZnO-NP-treated rats.
In the present study, morphometric measurement in lung sections illustrating the mean values of area % of Ki67 and caspase-3-immunopositive inflammatory cells, bronchiolar and alveolar epithelium, recorded a significant increase in the NP group and NP + VC group in comparison to the control group, while a significant decline was demonstrated in the NP + VC group when compared to the NP group.
Going with these results, it was previously demonstrated that ZnO-NPs cause elevation in Ki67-immunopositive cells in lung tissue. Several studies have previously manifested that VC is beneficial for alleviating oxidative stress-induced lung injury preventing cytotoxicity and apoptosis.
The NP group spleen tissues observed in our study were stained in H and E and showed a loss of splenic structural pattern. Some deformed white pulps showed some distortion of lymphatic architecture, mononuclear inflammatory cell aggregations in red pulp infiltrating the splenic cords as well as dilated splenic sinuses. Brown deposits inside macrophages in red pulp were observed. The NP + VC group showed an almost normal appearance of white pulp. Some areas in the red pulp showed clusters of mononuclear inflammatory cells, slightly dilated splenic sinuses, and macrophages containing brown deposits.
In accordance with these results, recent experiments have shown in the red pulp of ZnO-NP-exposed spleen tissues, dispersed clusters of lymphocytes, congested blood vessels, and hemosiderin-laden cells which were described as splenic macrophages stimulated to eliminate damaged RBCs. Furthermore, Elshama et al., 2017, demonstrated dilatation of splenic sinuses of red pulp and destruction of white pulp in the spleens of ZnO-NP-treated rats. Similarly, Ibrahim et al., 2018, illustrated that the titanium dioxide NP-treated group showed marked disturbance in architecture of white pulp. It was reported that ZnO-NPs induced immunotoxicity in the spleen through oxidative and inflammatory pathways.
Parallel to our findings, Afshari-Kaveh et al., 2021, and Mihailovic et al., 2021, documented severe alterations in the oxidative status of rat spleen tissues treated with titanium dioxide NPs. However, treatment with Vitamins A and E demonstrated antioxidant properties which provided protection for the spleen tissue against to toxicity of NPs.
Ultrastructural examination of spleen tissue from the NP group in the present work showed congested splenic sinusoids filled with RBCs, degenerated lymphocytes with irregular outline and condensed chromatin, macrophages with irregular nuclei, disrupted rER organization, multiple lysosomes, cytoplasmic vacuoles, engulfed RBCs, and phagosomes containing electron-dense particles. Monocytes with cytoplasmic vacuolations were noticed. NP + VC sections of the spleen a partially preserved histological picture; however, some degenerative alterations were noticed such as macrophages with cytoplasmic vacuoles and occasional phagosomes containing electron-dense particles, reticular cells with hyperchromatic nuclei, and plasma cells with slight dilatations in rER cisternae. Occasional mast cells were encountered.
Going with our mentioned results, ultrastructure findings observed by Elshama et al., 2017, of spleen tissue of ZnO-NP-treated rats have demonstrated stasis of blood in splenic sinusoids in the red pulp and infiltration of macrophages and lymphocytes which had cytoplasmic vacuoles. Furthermore, plasma cells with hyperchromatic nuclei and dilatation in the cisterna of rER were detected by Ibrahim et al., 2018, in spleens of titanium dioxide NP-treated rats. Furthermore, Mazen et al., 2017, found in silver NP-intoxicated spleens, lymphocytes with condensed chromatin, and macrophages with phagosomes and engulfing RBCs or silver NPs.
It was previously reported that ZnO-NPs stimulate the marker of cell maturation (CD11b) needed for macrophages' activation which accentuates their part in immune activation. Macrophages responsible for uptake and metabolism of foreign particles are located principally in the liver, spleen, and lungs. It was reported that NPs can reach the spleen through either internalization in RBCs or transport by means of macrophages.
Morphometric measurement of mean values of area % of Ki67 and caspase-3-immunopositive cells in spleen sections of the current study further confirmed the histological picture. Significant increases were observed in the NP group and NP + VC group in comparison with the control group. Nonetheless, the NP + VC group displayed a significant decline when compared to the NP group.
Interestingly, the Ki67 antibody immunoreaction of the NP group was scattered throughout deformed white and also red pulps, while that of the NP + VC group was expressed mainly in the germinal centers in white pulps and less in marginal zone, which resembles the immunoexpression of control sections, in addition to few cellular clusters in red pulps.
Agreeing with these results, Hassanen et al., 2014, recorded a significantly elevated PCNA antibody immunoexpression in spleen tissues of chitosan-coated silver NP-intoxicated rats. They proposed that ROS generation could have initiated the cell cycle's progression.
Mazen et al., 2017, demonstrated a strong caspase-3-immunopositive cytoplasmic reaction in splenocytes of red and white pulps in sliver NP-intoxicated rats. Notably, Xiong et al., 2022, stated that caspase-3 is upregulated by exposure to metal-based NPs. Several recent reports have demonstrated that apoptosis triggered by ZnO-NPs was related to the activation of caspase-3.
| Conclusion|| |
ZnO-NPs cytotoxic effects were portrayed in the liver, lung, and spleen tissues of rats. The featured degenerative changes were assessed by biochemical, histological, immunohistochemical, and ultrastructural studies. Ascorbate “VC” has proven to provide a beneficial protective effect. Further studies exploring the various advantages of antioxidant, and the roles they play against systemic toxicity are recommended.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Yousef MI, Mutar TF, Kamel MA. Hepato-renal toxicity of oral sub-chronic exposure to aluminum oxide and/or zinc oxide nanoparticles in rats. Toxicol Rep 2019;6:336-46.
Liu J, Kang Y, Yin S, Song B, Wei L, Chen L, et al
. Zinc oxide nanoparticles induce toxic responses in human neuroblastoma SHSY5Y cells in a size-dependent manner. Int J Nanomed 2017;12:8085-99.
Dağlıoğlu Y, Yılmaz Öztürk B. Effect of concentration and exposure time of ZnO-TiO2 nanocomposite on photosynthetic pigment contents, ROS production ability, and bioaccumulation of freshwater algae (Desmodesmus multivariabilis). Caryologia 2018;71:13-23.
Mohd Yusof H, Abdul Rahman N, Mohamad R, Hasanah Zaidan U, Samsudin AA. Antibacterial potential of biosynthesized zinc oxide nanoparticles against poultry-associated foodborne pathogens: An in vitro
study. Animals 2021;11:2093.
Maheswaran H, Wong LS, Dhanapal AC, Narendhirakannan RT, Janakiraman AK, Djearamane S. Toxicity of zinc oxide nanoparticles on human skin dermal cells. J Exp Biol Agric Sci 2021;9:S95-100.
Jiang J, Pi J, Cai J. The advancing of zinc oxide nanoparticles for biomedical applications. Bioinorg Chem Appl 2018;2018:1062562.
Espitia PJ, Otoni CG, Soares NF. Zinc oxide nanoparticles for food packaging applications. In: Antimicrobial Food Packaging. Food and Bioprocess Technology 2012;5:1447-64.
Venis RA, Basu OD. Silver and zinc oxide nanoparticle disinfection in water treatment applications: Synergy and water quality influences. H2Open J 2021;4:114-28.
Anjum S, Hashim M, Malik SA, Khan M, Lorenzo JM, Abbasi BH, et al
. Recent advances in zinc oxide nanoparticles (ZnO NPs) for cancer diagnosis, target drug delivery, and treatment. Cancers (Basel) 2021;13:4570.
Pinho AR, Martins F, Costa ME, Senos AM, Silva OA, Pereira ML, et al
. In vitro
cytotoxicity effects of zinc oxide nanoparticles on spermatogonia cells. Cells 2020;9:1081.
Scherzad A, Meyer T, Kleinsasser N, Hackenberg S. Molecular mechanisms of zinc oxide nanoparticle-induced genotoxicity short running title: Genotoxicity of ZnO NPs. Materials (Basel) 2017;10:1427.
Hegazy AA, Ahmed MM, Shehata MA, Abdelfattah MM. Changes in rats' liver structure induced by zinc oxide nanoparticles and the possible protective role of vitamin E. Int J Hum Anat 2018;1:1-16.
Chen A, Feng X, Sun T, Zhang Y, An S, Shao L. Evaluation of the effect of time on the distribution of zinc oxide nanoparticles in tissues of rats and mice: A systematic review. IET Nanobiotechnol 2016;10:97.
Sequeira IR. Higher doses of ascorbic acid may have the potential to promote nutrient delivery via intestinal paracellular absorption. World J Gastroenterol 2021;27:6750-6.
Caritá AC, Fonseca-Santos B, Shultz JD, Michniak-Kohn B, Chorilli M, Leonardi GR. Vitamin C: One compound, several uses. Advances for delivery, efficiency and stability. Nanomed Nanotechnol Biol Med 2020;24:102117.
Mihailovic V, Katanic Stankovic JS, Selakovic D, Rosic G. An overview of the beneficial role of antioxidants in the treatment of nanoparticle-induced toxicities. Oxid Med Cell Longev 2021;2021:7244677.
Espanani HR, Faghfoori Z, Izadpanah M, YousefiBabadi V. Toxic effect of nano-zinc oxide. Bratisl Lek Listy 2015;116:616-20.
Mahamuni PP, Patil PM, Dhanavade MJ, Badiger MV, Shadija PG, Lokhande AC, et al
. Synthesis and characterization of zinc oxide nanoparticles by using polyol chemistry for their antimicrobial and antibiofilm activity. Biochem Biophys Rep 2019;17:71-80.
Somayeh B, Mohammad F. Vitamin C can reduce toxic effects of Nano Zinc Oxide. Int Res J Biol Sci 2014;3:2278-3202.
Taghyan SA, Messiry HE, Zainy MAE. Evaluation of the toxic effect of silver nanoparticles and the possible protective effect of ascorbic acid on the parotid glands of albino rats: An in vivo
study. Toxicol Ind Health 2020;36:446-53.
Hohlbaum K, Bert B, Dietze S, Palme R, Fink H, Thöne-Reineke C. Impact of repeated anesthesia with ketamine and xylazine on the well-being of C57BL/6JRj mice. PLoS One 2018;13:e0203559.
Osman A, Oze M, Afify SM, Hassan G, EL-Ghlban S, Nawara HM, et al
. Tumor-associated macrophages derived from cancer stem cells. Acta Histochem 2020;122:151628.
Gadd VL. Combining immunodetection with histochemical techniques: The effect of heat-induced antigen retrieval on picro-Sirius red staining. J Histochem Cytochem 2014;62:902-6.
Graham L, Orenstein JM. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nat Protoc 2007;2:2439.
Kim HY. Analysis of variance (ANOVA) comparing means of more than two groups. Restor Dent Endod 2014;39:74.
Islam F, Shohag S, Uddin MJ, Islam MR, Nafady MH, Akter A, et al
. Exploring the journey of zinc oxide nanoparticles (ZnO-NPs) toward biomedical applications. Materials (Basel) 2022;15:1-31.
Gaharwar US, Meena R, Rajamani P. Biodistribution, clearance and morphological alterations of intravenously administered iron oxide nanoparticles in male wistar rats. Int J Nanomed 2019;14:9677-92.
Sukhanova A, Bozrova S, Sokolov P, Berestovoy M, Karaulov A, Nabiev I. Dependence of Nanoparticle Toxicity on Their Physical and Chemical Properties n.d. Nanoscale Research Letters 2018;13:Article 44. Available from: https://doi.org/10.1186/s11671-018-2457-x
Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arab J Chem 2019;12:908-31.
Almansour MI, Alferah MA, Shraideh ZA, Jarrar BM. Zinc oxide nanoparticles hepatotoxicity: Histological and histochemical study. Environ Toxicol Pharmacol 2017;51:124-30.
Tang HQ, Xu M, Rong Q, Jin RW, Liu QJ, Li YL. The effect of ZnO nanoparticles on liver function in rats. Int J Nanomed 2016;11:4275.
Yousef M, Abuzreda A, Kamel M. Cardiotoxicity and lung toxicity in male rats induced by long-term exposure to iron oxide and silver nanoparticles. Exp Ther Med 2019;18:4329-39.
Yan G, Huang Y, Bu Q, Lv L, Deng P, Zhou J, et al
. Zinc oxide nanoparticles cause nephrotoxicity and kidney metabolism alterations in rats. J Environ Sci Health A Tox Hazard Subst Environ Eng 2012;47:577-88.
Slama IB. Sub-acute oral toxicity of zinc oxide nanoparticles in male rats. J Nanomed Nanotechnol 2015;6:3.
Abass MA, Selim SA, Selim AO, El-Shal AS, Gouda ZA. Effect of orally administered zinc oxide nanoparticles on albino rat thymus and spleen. IUBMB Life 2017;69:528-39.
Babu EP, Subastri A, Suyavaran A, Premkumar K, Sujatha V, Aristatile B, et al
. Size dependent uptake and hemolytic effect of zinc oxide nanoparticles on erythrocytes and biomedical potential of ZnO-ferulic acid conjugates. Sci Rep 2017;7:4203.
Manke A, Wang L, Rojanasakul Y. Mechanisms of nanoparticle-induced oxidative stress and toxicity. Biomed Res Int 2013;2013:942916.
Elshama SS, Salem RR, Osman HE, El-Kenawy AE. Toxic effect of sub-chronic use of zinc oxide nanoparticles on the lymphatic system of adult albino rats. Curr Top Toxicol 2017;13:127-37.
Fukui H, Iwahashi H, Endoh S, Nishio K, Yoshida Y, Hagihara Y, et al
. Ascorbic acid attenuates acute pulmonary oxidative stress and inflammation caused by zinc oxide nanoparticles. J Occup Health 2015;57:118-25.
Almansour M, Sajti L, Melhim W, Jarrar B. Ultrastructural hepatic alterations induced by 35 nm zinc oxide nanoparticles. Nanosci Nanotechnol Lett 2015;7:763-9.
Al-Ali AA, Al-Tamimi SQ, Al-Maliki SJ, Abdullah MA. Toxic effects of zinc oxide nanoparticles and histopathological and caspase-9 expression changes in the liver and lung tissues of male mice model. Appl Nanosci 2022;12:193-203.
Al-Rasheed NM, Al-Rasheed NM, Abdel Baky NA, Faddah LM, Fatani AJ, Hasan IH, et al.
Prophylactic role of a-lipoic acid and vitamin E against zinc oxide nanoparticles induced metabolic and immune disorders in rat's liver. Eur Rev Med Pharmacol Sci 2014;18:1813-28.
Fattin SM, ElSalam NF, Bahaa N, Baher W. Effect of silica oxide nanoparticles on liver of adult male albino rat. Light and electron microscopic study. Egypt J Histol 2017;40:345-61.
Yu Z, Li Q, Wang J, Yu Y, Wang Y, Zhou Q, et al
. Reactive oxygen species-related nanoparticle toxicity in the biomedical field. Nanoscale Res Lett 2020;15:1-14.
Bayat M, Daei S, Ziamajidi N, Abbasalipourkabir R, Nourian A. The protective effects of vitamins A, C, and E on zinc oxide nanoparticles (ZnO NPs)-induced liver oxidative stress in male Wistar rats. Drug Chem Toxicol 2021:1-10.
Abu-Dief EE, Abdel-Aziz HO, Nor-Eldin EK, Khalil KM, Ragab EE. Ultrastructural, histochemical and biochemical effects of titanium dioxide nanoparticles on adult male albino rat liver and possible prophylactic effects of milk thistle seeds. Egypt J Histol 2018;41:1-10.
Shamel M, Riad D, Al Ankily M. Histological and ultrastructural study of silver nanoparticles toxicity and the possible protective effect of vitamin C on submandibular salivary glands of albino rats. Int J Dent Oral Sci 2021;8:2166-71.
Kaźmierczak-Barańska J, Boguszewska K, Adamus-Grabicka A, Karwowski BT. Two faces of vitamin C-antioxidative and pro-oxidative agent. Nutrients 2020;12:1501.
Wesselkamper SC, Chen LC, Kleeberger SR, Gordon T. Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice. Am J Physiol Lung Cell Mol Physiol 2001;281:L1200.
Mohamed MW, El-Fakharany YM, Hassan NM, Elsayed HM. The role of ascorbic acid in zinc oxide nano-particles induced lung toxicity in adult male albino rats. Egypt J Forensic Sci Appl Toxicol 2019;16:35-55.
Mohammed HL, El Shakaa N, Bahaa N, Zeid AA. A histological study on the acute effect of zinc oxide nanoparticles administered by different routes on albino rat lung. J Microsc Ultrastruct 2021;10:72-80.
Elbakary R, Okasha E, Hassan Ragab A, Ragab M. Histological effects of gold nanoparticles on the lung tissue of adult male albino rats. J Microsc Ultrastruct 2018;6:116. [Full text]
Ibrahim R, Salem MY, Helal OK, Abd El-Monem SN. Effect of titanium dioxide nanoparticles on the spleen of adult male albino rats: Histological and immunohistochemical study. Egypt J Histol 2018;41:311-28.
Afshari-Kaveh M, Abbasalipourkabir R, Nourian A, Ziamajidi N. The protective effects of vitamins A and E on titanium dioxide nanoparticles (nTiO2)-induced oxidative stress in the spleen tissues of male wistar rats. Biol Trace Elem Res 2021;199:3677-87.
Mazen NF, Saleh EZ, Mahmoud AA, Shaalan AA. Histological and immunohistochemical study on the potential toxicity of sliver nanoparticles on the structure of the spleen in adult male albino rats. Egypt J Histol 2017;40:374-87.
Shi H, Magaye R, Castranova V, Zhao J. Titanium dioxide nanoparticles: A review of current toxicological data. Part Fibre Toxicol 2013;10:15.
Hassanen EI, Khalaf AA, Tohamy AF, Mohammed ER, Farroh KY. Toxicopathological and immunological studies on different concentrations of chitosan-coated silver nanoparticles in rats. Int J Nanomed 2019;14:4723-39.
Department of Histology, Faculty of Medicine, Kafrelsheikh and Horus University, Kafrelsheikh 33511
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]