Toxicity assessment of copper oxide nanoparticles: In vivo study

Bashir Jarrar; Mansour Almansour; Qais Jarrar; Amin Al-Doaiss; Shiou Yih Lee; Djalila Boudemagh

doi:10.1515/ntrev-2024-0122

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Toxicity assessment of copper oxide nanoparticles: In vivo study

Bashir Jarrar , Mansour Almansour , Qais Jarrar , Amin Al-Doaiss , Shiou Yih Lee and Djalila Boudemagh

Published/Copyright: November 25, 2024

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From the journal Nanotechnology Reviews Volume 13 Issue 1

Abstact

Copper oxide nanoparticles (CuO NPs) have wide range of application in many fields of industry, agriculture, cosmetics, and health care with probable risk to human health. The present study was conducted to find the chronic toxicity of these nanoparticles in the vital organs. Male healthy Wister Albino rats were subjected on daily bases to 35 intraperitoneal administration of 25 nm CuO NPs (2 mg/kg). All animals were subjected to morphological, histological, histochemical, and ultrastructural examinations. Exposure to CuO NPs induced reduction in body weight gain, urine retention, back arching, and renal calculi formation. The renal tissues demonstrated tubular hydropic degeneration, glomerular hypercellularity, blood vessels congestion and dilatation, renal interstitial oedema, mitochondrial injury, and lysosomal hypertrophy. Conversely, the liver displayed hepatocyte insultation, sinusoidal dilatation, Kupffer cells hyperplasia, inflammatory cell infiltration, hepatocytes mitochondrial cristolysis, mitochondrial swelling, lysosomal hyperplasia, and nuclear alterations. Furthermore, the cardiac tissues demonstrated congestion, cardiocytes disarray, and disorganization. In addition, the neural tissue exhibited Purkinje cells degeneration and cerebral cortex spongiosis. The results suggest that CuO nanomaterials engage with the vital organs’ components, potentially leading to alterations that could affect the organs function. Further research is encouraged for better understanding the mechanisms involved in pathogenesis of CuO NPs.

Keywords: nano-CuO; vital organs; histochemical changes; ultrastructural injury; nanotoxicity

1 Introduction

Copper oxide nanoparticles (CuO NPs) have been utilized across various sectors, including antimicrobial agents, lubricants, anti-tumor, lubricants for textiles and metallic coatings, osteoporosis treatment, livestock feed additives, scientific and medical imaging, cosmetics, electronics, food industry, and drug delivery [1,2]. These nanomaterials have great catalytic activities and can be applied to biosensors and electrochemical sensors. The investment of CuO NPs and their nanocomposite is increasing with prediction of nano-copper global production to exceed 1,650 tons by 2025 [3].

Nano-copper can enter the body through various exposure routes including inhalation, dermal contact, and ingestion. Some investigations showed that kidney, spleen, and liver are the main target organs for the toxic CuO nanomaterials [4]. Other reports indicated that CuO NPs could decrease the plasma level of catalase, superoxide dismutase, and glutathione but increased malondialdehyde production [5,6]. Furthermore, CuO nanomaterials could cause histological changes such as apoptosis, inflammation, lipid and energy metabolism dysfunction, genotoxicity, oxidative stress, and mitochondrial damage [7–9]. CuO NPs produce reactive oxygen species (ROS) along with antioxidant defense system blockage leading to the oxidative damage and cell death [10]. In addition, CuO NPs can block the cell antioxidant defense [11]. However, the genotoxicity of CuO NPs is dependent upon several factors such as shape, charge, size, dose, and duration of exposure to these nanoparticles [12].

Studies revealed that liver and kidneys are targets for CuO NPs toxicity and subjection to these nanomaterials could cause marked degenerative changes in the renal tissues [3,4]. Moreover, treatment of Hep-2 cell line with copper oxide nanomaterials could induce oxidative damages to these cells and significantly increase of superoxide dismutase activity and level of thiobarbituric acid-reactive substances but reduce catalase activity [13,14]. Few studies explored the potential impacts of CuO NPs on the vital organs. Accordingly, the current investigation was conducted to find out the morphological, histological, histochemical, and ultrastructural alterations in the vital organs that might be induced by chronic exposure to 25 nm nano-CuO.

2 Materials and methods

2.1 Animals and conditions

Eighteen healthy adult male Wistar albino rats (Rattus norvegicus) were procured from Jerash University’s colony. These rats aged 16 weeks and had an average body weight ranging from 200 to 215 g. They were randomly divided into two groups: a control group and a CuO NPs-treated group, each comprising nine rats. Both groups of rats understudy a 7-day acclimatization period and were housed in conventional rat cages (measuring 48.0 × 26.5 × 21.0 cm) under controlled environmental conditions of 24 ± 1°C and 35% humidity, with a 12 h light/12 h dark cycle. The animals had continuous access to standard rodent laboratory diet provided by Jordan Feed Company (Amman, Jordan) and tap water sourced from Jerash University’s animal house.

2.2 CuO NPs

CuO NPs in the form of nanopowder were procured from Sigma (Aldrich, USA) for use in the current study. The manufacturer’s certificate provided the following specifications: a diameter of less than 50 nm, with approximately 82% falling within the range of 20 ± 5 nm and an average size of 25 nm. The nanoparticles also possessed a surface area of 29 m²/g, a boiling point of 2,567°C, and a metal content of 99.99%. The nanoparticle size was verified using transmission electron microscopy (TEM) technique.

2.3 Experimental protocol

A fresh suspension of CuO NPs was subjected to ultrasonication after being diluted in deionized water enriched with 0.1 mM sodium citrate. All NPs solutions were meticulously prepared to facilitate intraperitoneal (IP) administration, which is considered an optimal route for absorption into the circulatory system. These doses were administered in a volume of 0.4 mL. In the experimental group, each rat received 35 IP injections of CuO NPs at a dosage of 2 mg/kg, once per day, with injections given five times per week. The control group rats received a daily dose equivalent in volume to the vehicle solution, consisting of deionized water containing 0.1 mM sodium citrate, warmed to 37°C, using the same injection procedure. The choice of the dose (2 mg/kg) was based on previous in vivo studies investigating the toxicity of CuO NPs [15,16]. Before injection, each animal was securely restrained in a head-down position, with the injection site located in the lower right quadrant of the abdomen to prevent injury to internal organs.

After a 24 h interval following the last injection, the rats were anesthetized via the IP route using a cocktail of ketamine and xylazine at a dosage of 80/8 mg/kg, dissolved in normal saline. They were then dissected to extract vital organs for analysis. Throughout the study, all rats were closely monitored for their responses to CuO NPs exposure, overall health, body weight changes, morbidity, and mortality.

All animals under study were treated and all experimental works were practiced according to the guide of Canadian Council on Animal Care which is followed by Jerash University [17]. In addition, the ethical committee of Jerash University (reference number 3/1/2023/2024) approved the study protocol.

2.3.1 Morphometric observations

All animal understudies were observed for the mortality rate, general wellbeing, behavior patterns, food consumption, water intake, body weight gain, and organs indices. In addition, the treated rats’ body weight, average food consumption, and water intake were monitored weekly during the period of treatment. The chemical content of the demonstrated renal calculi was determined using a Fourier Transform Infrared spectrometer (FTIR; BRUKER Vertex 80, Switzerland) in a (5,000–500) cm⁻¹ wavenumber range.

2.3.2 Histological processing

Tissue samples were collected from the liver, kidney, heart, and brain of all the rats. These samples were initially fixed in a 10% neutral buffered formalin solution. Subsequently, they underwent a dehydration process with a series of gradually increasing concentrations of ethyl alcohol. Following dehydration, the samples were cleared using chloroform, impregnated, embedded, and encased in paraffin wax with a melting point of 58°C. The resulting histological sections, measuring 4–5 µm in thickness, were then subjected to staining with both hematoxylin and eosin (H&E) as well as Mallory trichrome stains [18].

2.3.3 Histochemical processing

Histological sections from all the rats, prepared in parallel with paraffin embedding, were subjected to the histochemical stain: the Periodic acid-Schiff (PAS) reaction, Masson trichrome, and methyl bromophenol blue stain [19].

2.3.4 TEM processing

Tissue blocks from the liver and kidneys of two rats from each group were used to find out the hepatic and renal tissues ultrastructural alterations according to the protocol described by Hayat [20].

2.4 Statistical analysis

Data from the morphometric investigation underwent rigorous statistical analysis, employing student’s t-test. Statistical significance was determined with a threshold of P values <0.05. The analysis was conducted using the Statistical Package for the Social Sciences software (IBM Analytics in the USA).

3 Results

3.1 Body weight gain alteration

After subjection to 35 IP injections of CuO NPs, a change in the body weight gain was demonstrated (Table 1). In comparison with control rats, the treated ones showed nonsignificant body weight gain (P value = 0.068). Control rats had a significant normal weight gain (P value = 0.035) during the treatment period.

Table 1

Alterations on the average body weight (g) of rats exposed to 35 injections of CuO NPs

Groups	Starting weight	Weight after 35 injections of CuO NPs	P-value
Control rats	179 ± 12	201 ± 17	0.035*
CuO NPs-treated rats	182 ± 15	191 ± 8	0.068

*Indicates statistically significant (P-value ≤ 0.05) under student’s t-test.

3.2 Morphometric alterations

No mortality occurred in CuO NPs-treated rats during the study. However, CuO NPs-treated rats exhibited abdominal ballooning, back arching, nose oozing of pinkish fluid during injection time, testicular enlargement, bloody urination, and urinary bladder congestion. In addition, gross examination with the bare eye and dissecting lenses of the internal organs of each dissected rat under study demonstrated that CuO NPs-treated rats showed lung abscess mainly in the left lung, seminal vesicles enlargement returned to normal after urinary bladder ruptured and urinary bladder ballooning.

Eight out of 18 (44.4%) rats that were exposed to CuO NPs demonstrated renal calculi (Figure 1). The largest calculus had a weight of 1.27 g with the smallest of 1.13 g. The average weight of the control kidney was 1.38 g. FTIR analysis confirmed that the renal calculi contained a mixture of calcium phosphate, uric acid, calcium oxalate monohydrate, and cystine.

Figure 1

Light photographs of renal calculi induced in rats subjected to 35 injections of CuO NPs.

3.3 Histological alterations

The following histological, histochemical, and ultrastructural alterations were detected in all rats exposed to CuO NPs.

3.3.1 Renal histological alterations

Compared with the control kidneys, the renal tissues of the rats that are subjected to 25 nm CuO NPs demonstrated inflammatory cells inflammation, glomerular atrophy, glomerular fragmentation, renal tubules necrosis, renal tubules hydropic degeneration, fibrocytes proliferation, hyaline casts formation, renal oedema, and renal congestion (Figure 2a–j). No indication of glomerular or interstitial fibrosis was detected.

Figure 2

H&E stained sections in the kidneys demonstrating (a) control animal renal tissue, ×160. Kidney sections of CuO NPs-treated rats showing: (b) inflammatory cells inflammation (arrow), ×160; (c) glomerular atrophy (arrow), ×160; (d) glomerular fragmentation (arrow), ×160; (e) tubules necrosis, ×320; (f) tubules hydropic degeneration (triple headed arrow), ×480; (g) fibrocytes proliferation (arrows), ×160; (h) hyaline casts precipitation (arrows), ×160; (i) interstitial edema (star), ×160; and (j) renal congestion (arrow), ×160.

3.3.2 Renal cells ultrastructural changes

The renal cells of animals subjected to CuO NPs demonstrated glomerular alterations and mitochondrial cristolysis and swelling, cytoplasmic vacuolization, lysosomal activity, apoptosis, dilatation of the endoplasmic reticulum, nuclear deformity, chromatin dissolution, and thickening of the basement membrane (Figure 3a and b).

Figure 3

TEM micrographs of renal cells from (a) control rat showing normal euchromatin, nuclear envelop consistency (arrow) with prominent normal mitochondria (cloud, ×8,000). (b) CuO NPs-treated rat showing mitochondrial crystolysis and swelling (arrow-left right), chromatin dissolution (star) basement membrane thickening (arrow), and lysosomal hypertrophy, ×8,000.

3.3.3 Hepatic histological alterations

Chronic subjection to CuO NPs caused the following histological hepatic alterations: hepatocytes hydropic degeneration, sinusoidal dilatation, Kupffer cells hyperplasia, and inflammatory cells infiltration (Figure 4a–e). No hepatic fibrosis or cirrhosis were detected in the liver of all CuO NPs-treated rats.

Figure 4

Light micrographs of hepatic tissue stained by H&E stain showing (a) control rat liver, ×180. Sections in the hepatic tissue of CuO NPs-treated animals demonstrating: (b) hydropic degeneration (arrows), ×400; (c) sinusoidal dilatation, ×180; (d) Kupffer cells enlargement, ×180; and (e) inflammatory cells infiltration (arrow), ×128.

3.3.4 Hepatocytes ultrastructural alterations

The hepatic cells of nano-copper-treated rats demonstrated mitochondrial crystolysis, swelling and matrix lysis, lysosomal hyperplasia, fat droplets precipitation, phagocytized bodies, and nuclear envelope irregularity (Figure 5a and b).

Figure 5

TEM micrographs of hepatic cells from (a) control rat showing normal euchromatin with numerous mitochondria. Note that the nuclear envelope is consistent (white arrow), ×5,000. (b) CuO NPs-treated rat demonstrating mitochondrial (m) swelling (stars), crystolysis and matrix lysis, fat droplets (cloud), and nuclear envelope irregularity (white arrow: up), ×20,000.

3.3.5 Hepatocytes glycogen depletion

By investing PAS stain, the hepatocytes of CuO NPs-treated rats showed depletion of glycogen mainly in the degenerative hepatic cells (Figure 6a and b).

Figure 6

Light micrographs of sections stained with PAS reaction in the hepatic tissue showing: (a) control rat hepatic cell with normal glycogen content, ×128 and (b) CuO NPs-treated rats showing partial glycogen depletion, ×128.

3.3.6 Cardiac histological alterations

The cardiac tissue of the CuO NPs-treated rats that were subjected to 35 IP injections showed cardiac congestion, cardiocytes disarray and disorganization, and occasional hydropic degeneration (Figure 7a–d). No cardiac fibrosis was observed in the heart of all rats under study.

Figure 7

Light micrographs of H&E-stained demonstrating: (a) control rat cardiac tissues, ×320; (b) cardiac congestion (star), CuO NPs-treated rats, ×180; (c) cardiocytes disarray and fibers disorganization, CuO NPs-treated rats, ×180; and (d) occasional hydropic degeneration (arrow), ×320.

3.3.7 Neural histological alterations

In comparison with the neural tissue of the control brains, the brain of CuO NPs-treated rats demonstrated necrotic Purkinje cells, occasional cerebral cortex spongiosis characterized by neural loss, and swelling (Figure 8a–c).

Figure 8

Light micrograph sections in the brain of (a) control rat cerebral cortex with normal pyramidal cells (arrows), normal glial cells (triple heads arrow), and normal neuropil (stars), H&E stain ×180. Brain sections of CuO NPs-treated rats showing: (b) necrotic Purkinje cells with normal granular cell layer (arrows), H&E stain ×320 and (c) neural spongiosis (arrow), methyl bromophenol blue stain, ×400.

4 Discussion

The results of the present study indicate that exposure to CuO nanomaterials could cause body weight gain reduction. This might be related to the impacts of nano-copper on the oxidative metabolic enzymes [21]. Moreover, back arching among CuO NPs-treated rats may indicate signs of pain associated with contraction of the abdominal muscles and abdominal pressing [22–24]. The seen urine retention might be associated to urethra blockage by precipitate from the formed calculi or from sloughing the renal epithelial lining. Furthermore, the FTIR analysis indicated that the induced calculi were devoid of copper compounds suggesting that the calculi formation resulted from disturbance on the body metabolism. Several reports demonstrated that the formation of renal calculi involves multiple metabolism-related factors [25]. Literature is devoid of any indications for renal calculi formation due to any kind of nanoparticles or nanostructures.

It is revealed that chronic exposure to CuO nanomaterials induced degenerative alterations in the renal cells, glomeruli, and renal interstitial tissues of the rats. This might be resulted from the interaction of CuO NPs with renal cell proteins forming corona [26]. In addition, the exhibited renal cells hydropic degeneration might be resulted from intracellular and interstitial fluid imbalance resulted from subjection to CuO NPs [27]. Moreover, the renal tissues showed degenerative changes mainly in the proximal convoluted tubules. This injury may indicate the greater viability of CuO NPs in the proximal tubules which are the primary sites of filtrate reabsorption. Furthermore, CuO NPs induced damage in the glomeruli and hyaline casts formation that may affect ions pump across the renal tubules [28].

Nano-copper induced liver injury indicating hepatic tissue susceptible to CuO NPs toxicity affecting liver function as site of detoxification [29]. The induced Kupffer cells hyperplasia may indicate that these nanomaterials could activate phagocytosis as a defense tool for detoxification [30]. Moreover, the sinusoidal dilatation may indicate sinusoids endothelia injury. In addition, the hepatocytes demonstrated partial glycogen depletion indicating an impact of CuO NPs on glycogenesis and glycolysis.

The renal and hepatic cells of animals subjected to CuO nanomaterials exposure show inflammatory cells. This injury may suggest the ability of these nanomaterials to interact with the hepatic and renal tissue components. Nanoparticles including CuO ones have almost similar measurements of the cellular macromolecules with possible interaction with these structures generating free radicals [31,32].

The induced cardiac alterations may reveal that cardic muscle fibers are affected by subjection to CuO NPs. The induced cardiocytes disoragnization may reveal cardiac degeneration, indicating cardiomyopathy [33,34]. Furthermore, the induced cardiocytes disarray might be associated with heart rate reduction [35].

Purkinje cells are specific to cerebellar cortex, play an essential role in movement coordination, cognition, and emotion [36]. Subjection to CuO NPs induced Purkinje cells degeneration indicating neurodegenerative cerebellar disorder [37]. Moreover, the seen spongiform degeneration in the cerebral cortex indicates brain atrophy associated with dementia resulted from loss of connection between neurons [38]. These alterations suggest an effect to CuO nanomaterials on the structure and function of the brain.

Collectively, CuO nanomaterials are found to be toxic and can cause morphological, histochemical, and histological changes in the ultrastructure of the vital organs. These results are congruent to some previous studies [39,40]. The induced injury by nano-copper might be associated to the ability of these nanomaterials to have long circulating residue with the bioavailability of the reactive surface area of these nanoparticles and high dissolution leading for intracellular ROS generation [41,42].

5 Conclusion

CuO nanomaterials could induce hepatic, renal, cardiac, and neural changes that may alter the structure and physiology of these vital organs. These alterations might be resulted from the induced oxidative stress and variable toxicokinetics by CuO NPs, causing injury to the vital organ tissues. It is suggested that there is a need to put more efforts to find out the pathogenesis and toxicity of the nano-copper materials on human and the ecosystems.

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Acknowledgments

The authors are grateful to Jerash University and Isra University for putting lab facilities under their disposal.

Funding information: The present project was supported by researchers supporting project number (RSPD2024R900) King Saud University, Riyadh, Saudi Arabia and by the Deanship of Scientific Research at King Khalid University, Abha, KSA through the research project (Grant number R.G.P.2/8/45).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Conflict of interest: The authors state no conflict of interest.
Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2024-08-11

Revised: 2024-09-28

Accepted: 2024-10-24

Published Online: 2024-11-25

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/ntrev-2024-0122

Keywords for this article

nano-CuO; vital organs; histochemical changes; ultrastructural injury; nanotoxicity

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