Effects of intraperitoneally injected silver nanoparticles on histological structures and blood parameters in the albino rat
Osama Mohamed M Sarhan,1,2 Rehab M Hussein3
1Department of Zoology, Faculty of Sciences, Fayoum University, Al Fayoum, Egypt; 2Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah Al-Mukarramah, Saudi Arabia; 3Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt
Background: The purpose of this study was to investigate the effect of acute dosing with silver nanoparticles (AgNPs) and identify potential ultrastructural alterations in the liver and kidney and their effect on blood parameters in the albino rat.
Methods: Twenty rats were used to assess the acute effects of AgNPs. Rats in the treatment group were injected intraperitoneally with 0.5 mL of distilled water containing AgNPs at a dose of 2,000 mg/kg body weight followed by a second injection after 48 hours. Control rats received two 0.5 mL doses of distilled water only. After 3 days, blood samples were collected, and the rat kidneys and livers were extracted and processed for electron microscopy to investigate for hematologic and histopathologic alterations.
Results: Renal tubules showed swollen epithelium with cytoplasmic vacuolization, thickening of the basement membrane, and destruction of some mitochondrial cristae. Podocytes showed elongation and swelling of their primary and secondary processes. The basement membrane of the capillary tufts became thicker. The hepatic tissue showed narrowing of the sinusoids, swollen hepatocytes with hypertrophied nucleoli, and accumulation of fat globules in the nucleoplasm and cytoplasm. The hepatic sinusoids showed hypertrophied endothelial and Kupffer. Destructed cristae of some mitochondria, endosomes, and larger lysosomes filled with Ag-NPs were also observed in the Kupffer cells. Significant increases were observed in white blood cell count, lymphocyte count, granulocytes, and hemoglobin. There was a significant increase in serum creatinine, urea, and aspartate and alanine aminotransferases.
Conclusion: To the best of the authors' knowledge, the ultrastructural changes in renal and liver tissue observed in this study have not been described before. Our results suggest that injection of AgNPs could have severe cytotoxic effects on the structure and function of these organs.
Keywords: silver nanoparticles, kidney, liver, blood, toxicity, rat
Nanoparticles are engineered materials produced within the nanoscale range of 1–100 nm in one or more dimensions.1 Pure silver has the highest electrical and thermal conductivity of all the metals and has low contact resistance. Silver nanoparticles (AgNPs) have unique physical and chemical properties2 and have been used in a wide variety of applications.3 AgNPs are the best known nanoproducts,4 have attracted considerable attention as antimicrobial agents,5 and have been incorporated into a number of products, including catheters, clothing, and electrical home appliances due to their high specific surface area and high proportion of surface atoms.6 More utilities were added to numerous consumer products including industrial and food products in addition to biological and health and medical applications.7,8
Nanosilver with a particle size less than 20 nm in diameter has been reported to be effective in the treatment of certain infectious diseases,9 and is effective in retarding growth of bacteria, mold, and harmful spores. It has been reported that silver is re-emerging as a viable treatment option for infections associated with burns, open wounds, and chronic ulcers.10 AgNPs can be ingested directly via water, food, cosmetics, drugs, and drug delivery devices.11 Some investigators have demonstrated that silver ions released from ingested products into the blood can accumulate in body organs and have toxic effects, especially in the liver and kidney.12 However, acute oral or transdermal doses of AgNPs (2,000 mg/kg body weight) in rats, guinea pigs, and rabbits have not resulted in significant clinical signs, mortality, acute irritation, or corrosive reactions affecting the eyes and skin.13 It has been reported that AgNPs are more toxic than other metal nanoparticles, including aluminum, iron, nickel, and manganese,14 but the mechanism of their toxicity is not clear.15
The aim of this study was to assess the biological risks and benefits of AgNPs. We studied the effect of acute dosing with AgNPs in order to identify potential ultrastructural alterations in the liver and kidney and blood parameters in the albino rat, in the hope of shedding light on the biological responses induced by acute dosing, and to assess cellular responses when these nanoparticles are used in biomedical applications.
Materials and methods
Silver nanopowder with a particle size less than 100 nm and a 99.9% trace metals basis was purchased from Sigma-Aldrich Chemicals, Cairo, Egypt. AgNPs have been dissolved in 0.5% aqueous carboxymethylcellulose (Sigma-Aldrich) were coated with carbon, mounted on an electron microscope grid (200 mesh),16 and visualized using a transmission electron microscope ([TEM]; JEM-100CXII, JEOL Ltd., Tokyo, Japan) operating at 80 kV. However, AgNPS in the injected doses should be distributed more uniformly by sonication for 10 minutes just before injection, to be taken by systemic circulation.
We purchased 20, 6-week-old male albino rats (weighing 260±5 g) from the animal house of Assiut University, Assiut, Egypt. The rats were randomly assigned to polycarbonate cages (five rats per cage), and acclimatized for 10 days at a temperature of 22°C±0.05°C and 51%±0.5% humidity with a 12/12 hour-light/dark cycle and access ad libitum to fresh tap water and a rodent diet for 2 weeks before the experiment. The experiments were performed in accordance with the research protocols established by the animal care committee of the National Research Center, Egypt.
Administration of AgNPs
Twenty rats were randomly allocated into two groups. Five rats were used as controls and injected intraperitoneally with 0.5 mL of sterile saline solution followed by a second dose after 48 hours. The remaining 15 rats were injected intraperitoneally with AgNPs at a dose of 2,000 mg/kg body weight dissolved in 0.5 mL of distilled water followed by a second injection after 48 hours.13 The rats were euthanized 3 days after the second injection, two blood samples were collected from the left ventricle of each rat under sterile conditions in heparinized tubes for hematology and non-heparinized tubes for biochemistry.
The blood samples were taken for a complete blood count, including erythrocytes, total leukocytes, hemoglobin, and hematocrit. Serum from the second blood sample was harvested after centrifugation at 5,000 rpm and stored at 20°C until determination of aspartate and alanine aminotransferases, as well as creatinine and urea according to standard methods.17,18
Using 2.5% glutaraldehyde, small slices of kidney and liver tissue were extracted, fixed immediately, rinsed in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed for 2 hours in 1% osmium tetroxide. Then they were rewashed with fresh sodium cacodylate buffer and dehydrated in an ascending series of ethanol. The specimens were embedded in Epon 812.19 Semithin (0.5–1 μm thickness) and ultrathin (700–800 A° thickness) sections were cut using an LKB ultramicrotome (LKB Produkter AB, Ltd., Stockholm, Sweden). The semithin sections were stained with toluidine blue and the ultrathin sections with uranyl acetate and lead citrate. The stained sections were then examined using the JEM-100CXII operating at 80 kV.
TEM imaging of AgNPs ranging from 20 nm to 65 nm in diameter was performed to confirm primary particle size and general morphology. Figures 1 and 2A show aggregation of AgNPs in the size range of 47–65 nm. However, smaller nanomolecules (less than 30 nm) appeared to exist as solitary entities (Figure 2B–D).
Figure 1 Transmission electron micrographs on supportive film showing the morphology of silver nanoparticles used in the experiment and indicating variable dimensions. Scale bars 61.5 and 64.5 nm.
Figure 2 A to D transmission electron micrographs on supportive film showing the morphology of silver nanoparticles used in the experiment and indicating variable dimensions. Scale bars from 25.1–58.3 nm.
Animal symptoms, food consumption, and general activity
No mortality, gross effects, or significant differences in food consumption or body weight were observed during the study period in any of the rats administered AgNPs when compared with the control group. However, treated rats showed a marked decrease in activity.
The treated animals showed distinct morphological changes in the kidney and liver on microscopic observation when compared with the control group, indicating unhealthy cells.
Figure 3 shows the normal histological structure of renal tubules in kidney tissue from the control group. However, the renal cortex in kidney tissue from the treated group shows swollen epithelium and cytoplasm containing numerous membranous vacuoles, with some nuclei showing hypertrophied nucleoli. Figure 4 shows a semithin section of kidney tissue from the treated group, with swelling of the tubular epithelium, cytoplasmic vacuolization, glomeruli with increased cellularity, and obliteration of Bowman’s space.
Figure 3 Semithin section of the kidney tubule tissue from the control group showing normal appearance of the tubular epithelium. Scale bar 50 μm.
Ultrastructural investigations of renal epithelium from control kidney tissue showed a normal brush border, basement membrane, and intact cell organelles, including mitochondria, nuclei, and a few membranous vacuoles (Figure 5). The glomeruli showed normal mesangial cells and a normal basement membrane in the capillary tufts, with some red blood cells in their lumen and normal surrounding primary and secondary processes of intact podocytes (Figure 6). In treated rats, the renal epithelium had a thickened basement membrane, and the cytoplasm showed some mitochondria with destroyed cristae and numerous large membranous vesicles (Figures 7 and 8). The glomeruli showed podocytes with swollen and elongated primary and secondary processes, and the basement membrane of the endothelial cells in the capillary tufts was thickened (Figure 9).
Semithin sections from a sample of control liver tissue demonstrated a normal histological structure (Figure 10), while semithin sections of treated liver tissue showed narrowing of the sinusoidal lumen and damaged hepatocytes (Figure 11). Ultrathin sections of treated liver tissue showed swollen hepatic cells, narrowing of the sinusoidal lumen, and appearance of hypertrophied Kupffer cells (Figure 12).
Figure 11 Semithin section of liver tissue from the treated group showing narrowing of the sinusoidal lumen. Scale bar 50 μm.
Ultrathin sections from control liver tissue showed normal hepatocytes on TEM. The nuclei had a normal ultrastructural appearance with a distinct nuclear envelope, and the nucleoplasm showed aggregations of euchromatin and heterochromatin granules (Figure 13). Other intact cytoplasmic organelles could be seen, including spheroid or ovoid mitochondria with well-developed cristae and flattened cisternae of rough endoplasmic reticulum studded with ribosomes; in addition, considerable numbers of glycogen granules were observed in the cytosol (Figure 14). In contrast, marked cytopathological changes were seen in hepatocytes from the treated group. Figure 15 showed a hepatocyte with strong cytoplasmic vacuolization, numerous intranuclear and intracytoplasmic fat globules of various sizes that appeared swollen, with smaller stacks of fragmented rough endoplasmic reticulum cisternae. Numerous intracytoplasmic and intranuclear fat globules of various sizes were intermingled with mitochondria that appeared swollen with obviously condensed electron-dense matrices and some of them without cristae (Figures 15 and 16). Some Kupffer cells showed numerous membranous vacuoles, fragmented rough endoplasmic reticulum cisternae, endosomes, and a large number of lysosomes filled with AgNPs of different sizes that appear as electron-dense material (Figures 17–19). Disse’s spaces contained fragmented microvilli (Figures 18, 20, and 21), and the mitochondria showed complete loss of internal ridges and matrices (Figures 22 and 23). Figure 23 shows degenerated hepatic Kupffer cells and nuclei, with destruction of the microvilli in Disse’s spaces and lysosomes filled with AgNPs that appear as electron-dense material.
Hematology and biochemistry
Complete blood count sampling in the treated group showed a significant increase in white blood cells and hemoglobin, with slight changes in red blood cell and hematocrit values (Table 1). In comparison with the control group, serum alanine and aspartate aminotransferase levels in the treated group were significantly elevated in comparison with those in the control group (Figure 24A and B), indicating disruption of liver function. Serum creatinine and urea were significantly increased, indicating changes in kidney function (Figure 24C and D).
AgNPs have received much attention due to their antimicrobial properties20,21 and their potential for application in the treatment of diseases that need a constant drug concentration in the blood or targeting of specific tissue.22–24 Despite these beneficial effects, some studies based on actual data from rat models suggesting that AgNPs may be cytotoxic even at low doses,25,26 potentially increasing reactive oxygen species,6,27 via which phospholipid membranes may be attacked, and decreasing function of the mitochondrial respiratory chain complexes in the liver, brain, and skeletal muscles.28,29 Nanoparticles released into the blood have been shown to accumulate, with toxic effects in the liver, kidney, and heart, causing scattered cytoplasmic vacuolization, appearance of chronic inflammatory cells, and congested and dilated blood vessels.12,30
The AgNPs used in our study appeared to be very uniform with a mostly spherical morphology and a particle size of 20–60 nm. Spherical, triangular, and hexagonal nanoparticles have better antimicrobial and physical properties if they are produced in a small size range.31 Jiang et al32 reported that AgNPs in the 40–50 nm size range demonstrated the best antimicrobial activity. However, sonification of the dose for 10 minutes immediately before injection may prevent aggregation of AgNPs before they can be taken up by the systemic circulation. The present work demonstrates that nonaggregated AgNPs may have increased hepatic and renal toxicity. Once AgNPs aggregate, significant loss of antibacterial activity occurs due to their inability to penetrate the plasma membrane and loss of surface area. (High School Nanoscience Program). Consequently, the decreased stability of AgNPs may lead to loss of their nanoscale properties.33 Further, aggregation of AgNPs decreased their effect and cellular uptake34 and modifies their bioavailability and toxicity.35
Our study suggests that the toxicity of AgNPs is dose-dependent and time-dependent, with more accumulation of silver in the liver than in the kidneys at 3 days post-injection. Limited tissue clearance was observed after 26 weeks in the liver, spleen, and lungs of mice treated with titanium dioxide nanoparticles.36 However, it has been concluded that the target organs for AgNP toxicity are the liver and kidneys of male and female rats and that deposition of AgNPs in tissue samples is size-dependent.16,37 It has also been demonstrated that different sized and shaped gold nanoparticles have different toxicity.38 Also, the smallest (mean size 2.4 nm) gold nanoparticles were found to be localized in the nucleus,39 and intermediate ones (size 5.5–8.2 nm) were partly delivered into the cytoplasm, causing greater oxidative stress and cytotoxic effects than larger nanoparticles,40 while larger gold nanoparticles (>16 nm) did not enter cells.39 In contrast, no histopathological evidence of tissue damage in response to treatment with gold or titanium dioxide nanoparticles has been observed in murine models,36,41 although the presence of these NPs inside intracellular vacuoles as endosomal containment.
The increased levels of silver in the kidney seen in our study are consistent with published literature showing that subchronic oral dosing with AgNPs induces minimal tubular basophilia in addition to unilateral or bilateral mineralization in the rat kidney.16 Other investigators have reported deposition of ingested silver in the renal tubules, the glomerular basement membrane,16,42–44 and mesangium,45 as well as proliferation of mesangial cells.46 Further, swelling of the renal epithelium and the presence of membranous vacuoles along with hypertrophied nucleoli have been demonstrated in all animals treated with AgNPs.44,47–50 Moreover, the present work suggests increasing glomerular cellularity and swelling of the primary and secondary processes of podocytes which, to the authors’ knowledge, has not been reported before.
With regard to liver toxicity, we found swelling of hepatocytes with prominent hypertrophied nucleoli and narrowing of the sinusoidal lumen. These results are consistent with previous reports of the liver being the target organ for AgNPs and the site at which they preferentially accumulate.11,16,44,51 The liver damage seen in the current study may be explained by: deposition of AgNPs in the hepatic Kupffer cells and endothelial cells lining the sinusoidal spaces;52 inhibition of the mitochondrial respiratory chain that normally produces energy for cells;4,28,53,54 generation of reactive oxygen species associated with inflammatory, oxidative, genotoxic, and cytotoxic events; and induction of apoptosis.29,51,53 Endocytosis of AgNPs by numerous endosomes and lysosomes in Kupffer cells was seen in the present work.
Similarly, our observation of intracellular fat globules in the cytoplasm of hepatocytes is new, although empty vacuole-like spaces have been reported in rats.44 Also, the first-pass effect of AgNPs in the liver resulted in excretion into the bile, inducing bile duct hyperplasia and focal, multifocal, or lobular necrosis.16 Silver had also been shown to be toxic to other organs, including the brain,55 and administration of gold nanoparticles has been associated with dilated interlobular sinusoidal capillaries and congestion of blood between hepatocytes in the liver.46 AgNPs endocytosed by Kupffer cells that appeared in the form of electron-dense materials were reported.43,50
With regard to the mitochondrial injury, this study confirms that the smallest Ag-NPs destructed biomembranes, decreased bioenergetics and adenosine triphosphate (ATP) levels which preceded cell death. Similarly, decreased ATP and creatine kinase levels suggest a potential for metabolic and cell cycle arrest, leading to widespread cell death.50,56
Complete blood counts revealed a significant increase in white cells and hemoglobin in the treated group, along with slight changes in red cell and hematocrit values. Moreover, when AgNPs are injected intravenously, they interact initially with the blood and its components and they may cause various immunogenic responses, inflammation, and changes in hematological parameters, including white cells and platelets.26 The changes in white and red blood cells reported here after the first injection of nanoparticles have been described before, and are possibly due to an increased immunogenic response26,57,58 or disturbances in signaling pathways and maturation of cells,59 which can affect red blood cells as well as the division and development of other cells.
With regard to biochemistry, significant elevations of serum alanine and aspartate aminotransferase, creatinine, and urea levels were seen, indicating disruptive changes in liver and kidney function. Previous studies have shown that metal nanoparticles alter the levels of various biochemical markers indicating changes in composition of serum enzyme levels,60 suggesting hepatocellular injury, hepatic inflammation, and impairment of kidney function.60,61
The aim of this study was to evaluate the potential toxicity of acute dosing with AgNPs. We found that acute doses of AgNPs were not associated with mortality and didn’t affect the normal activity (behavior) of examined rats. Renal damage was observed, including swollen epithelium in the renal tubules, cytoplasmic vacuolization, hypertrophied nucleoli, thickening of the basement membrane in the glomerular tufts, and reduction of Bowman’s space. On the other hand, liver tissue showed damaged hepatocytes and Kupffer cells with numerous intracellular and intranuclear fat globules, fragmented cisternae in the rough endoplasmic reticulum, and swollen or destroyed mitochondria with complete loss of internal ridges and matrices. It can be concluded that an acute dose of AgNPs will induce significant damage to the structure and function of the liver and kidney, as well as disrupting blood parameters. In this study, the acute dose generated remarkable toxic effect in killing any tumor cells. To protect healthy tissues and to reduce AgNPs toxicity, biodegradable, biocompatible organic substance, polymer matrix or even lipids should be used to encapsulate AgNPs. Further in vivo studies are essential to evaluate the critical concentration needed for AgNPs and to evaluate further ultrastructural effects on the high-energy consuming organs such as brain, skeletal muscles, and heart.
We are most grateful to Professor Ali Gab-Allah of the Faculty of Science at the University of Suez Canal in Egypt for his invaluable insights and suggestions. We also owe a tremendous amount of gratitude to Dr Abduraman A Noor, the CEO of Dr Sulub Leadership Center for his invaluable guidance.
The authors report no conflicts of interest in this work.
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