Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO Nanoparticles in Monosodium Urate Crystal-Induced Hyperuricemic BALB/c Mice

Article abstract of DOI:10.1007/s12011-021-02615-3

Nanotechnology is an intensive branch of science due to the unique features of nano range particles (1-100 nm). Their nano size results in a high surface area of absorption when orally administered. Monosodium urate crystal excessive deposition causes a commonly known inflammatory disease called gout into the synovial joints. Previously it has been observed that copper oxide nanoparticles (CuONPs) had a significant effect in reducing the serum uric acid levels in BALB/c mice as well as reducing the inflammation in the ankles of mice. This study was made to investigate the antioxidant and histopathological changes in hyperuricemic BALB/c mice upon the oral administration of copper oxide nanoparticles. Different concentrations of copper oxide nanoparticles 5, 10, and 20 ppm were given orally to gouty mice. To investigate the antioxidant activity of CuONPs, various antioxidant protocols were applied. It was noted that the nanoparticle-treated group of 20 ppm showed no significant changes in superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), and ROS values while the protein estimation values of the negative control group exhibited a significant increase (0.001). When compared to negative control, no significant effect was shown on the interpretation of histopathological changes of muscles, kidney, and liver tissues.

Full text of DOI:10.1007/s12011-021-02615-3

Biological Trace Element Research
https://doi.org/10.1007/s12011-021-02615-3
Evaluation of Antioxidant Activity and Histopathological Changes
Occurred by the Oral Ingestion of CuO Nanoparticles in Monosodium
Urate Crystal-Induced Hyperuricemic BALB/c Mice
Mubin Mustafa Kiyani1 & Maisra Azhar Butt2 & Hamza Rehman3 & Moheen Mustafa4 & Abdul Ghafoor Sajjad5
Syed Sajid Hussain Shah6 & Tariq Mahmood7 & Syed Ali Imran Bokhari3
&
Received: 11 September 2020 /Accepted: 27 January 2021
#
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Nanotechnology is an intensive branch of science due to the unique features of nano range particles (1-100 nm). Their nano size
results in a high surface area of absorption when orally administered. Monosodium urate crystal excessive deposition causes a
commonly known inflammatory disease called gout into the synovial joints. Previously it has been observed that copper oxide
nanoparticles (CuONPs) had a significant effect in reducing the serum uric acid levels in BALB/c mice as well as reducing the
inflammation in the ankles of mice. This study was made to investigate the antioxidant and histopathological changes in
hyperuricemic BALB/c mice upon the oral administration of copper oxide nanoparticles. Different concentrations of copper
oxide nanoparticles 5, 10, and 20 ppm were given orally to gouty mice. To investigate the antioxidant activity of CuONPs,
various antioxidant protocols were applied. It was noted that the nanoparticle-treated group of 20 ppm showed no significant
changes in superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), thiobarbituric acid reactive substances (TBARS),
and ROS values while the protein estimation values of the negative control group exhibited a significant increase (0.001). When
compared to negative control, no significant effect was shown on the interpretation of histopathological changes of muscles,
kidney, and liver tissues.
.
.
.
.
Keywords Antioxidant Copper Hyperuricemia Histopathology Nanoparticles
Introduction
In recent years, nanoparticle (NP) synthesis is considered the
most important area of research in the field of nanoscience and
technology [1]. NPs are playing essential roles in controlling
optical, chemical, physical, and electronic properties of
nanomaterials [2]. Generally, a particle size less than
100 nm is said to be in nano range [3, 4]. NPs are classified
based on their size, composition, shape, and origin. They have
high surface reactivity due to the large percentage of atoms
[5]. Because of this unique characteristic, they are unexpect-
edly reactive with biomolecules [6]. NPs have been extensive-
ly used in medical and pharmaceutical products such as tooth-
pastes, sunscreen lotions, shampoos, deodorants, and anti-
wrinkle creams [7]. Most of the cosmetics even are formulated
by using zinc oxide and titanium dioxide NPs [8]. NPs are also
reported as a beneficial agent in the field of biotechnology.
They are being used against as anti-cancerous, antifungal,
antibacterial, antioxidant, and anti-inflammatory agents. It is
reported that NPs are also used for targeted drug delivery due
to their bioavailability and bio reactivity [9].
* Hamza Rehman
hamzarehman51@gmail.com
1
Shifa College of Medical Technology, Shifa Tameer-e-Millat
University, Islamabad, Pakistan
2
Faculty of Rehabilitation and Allied Health Sciences, Riphah
International University, Islamabad, Pakistan
3
Department of Bioinformatics and Biotechnology, Faculty of Basic
and Applied Sciences, International Islamic University,
Islamabad, Pakistan
4
Faculty of Engineering, Federal Urdu University,
Islamabad, Pakistan
5
Department of Physical Therapy, Shifa Tameer-e-Millat University,
Islamabad, Pakistan
6
Department of Pathology, Northern Border University, Arar, Saudi
Arabia
7
Department of Nanoscience and Technology, National Center for
Physics, Islamabad, Pakistan

Kiyani et al.
In gouty arthritis, monosodium urate (MSU) crystals are
considered a key component associated with the rise of serum
urate content. Gout is characterized by inflammation due to
accumulation of MSU crystals within synovial fibroblast
which results in mechanical damage of synovial tissues.
During inflammation, tumor necrosis factor- α (TNF-α)
[10], chemokines (IL-8), invading neutrophils, local macro-
phages, and monocytes are responsible for the secretion of
pro-inflammatory mediators like interleukin-1β (IL-1β)
[11]. The MSU crystal initiates mast cell degradation that
induces inflammation which results in shearing and tearing
of adjacent synovial tissues [10].
incidence of gout (hyperuricemia) [20, 21]. Copper is also
known for its antagonistic effect in comparison with molyb-
denum. The active site of XOD and XDH is called as
molybdopterin. As copper concentration increases, the activi-
ty of these enzymes ceases [21]. Previous studies show that
copper is also used as an antibacterial agent [22]. It is required
for the antioxidant activity, carbohydrate, and xenobiotic me-
tabolism [23]. The xenobiotic metabolism was observed in
liver cells of rat when exposed to copper sulfate. It has been
observed that copper increases the expression of the genes
such as CYP1A1 and CYP1A2 which are involved in the
production of enzymes (cytochrome P450) responsible for
the xenobiotic activity. Copper is also well-known for its an-
tifungal [24] and anti-inflammatory activities. At the cellular
level, copper is considered an essential micronutrient for its
protein trafficking and redox property. A recent study has
proved the beneficial effects of copper in neurodegenerative
diseases. The cellular prion protein (PrPC), which is mostly
known as the neurodegenerative protein, is implicated in var-
ious biochemical processes, including neuritogenesis. The
role of PrPC neuritogenesis is impaired by N copper terminal
amino acids directly [25].
CuO-NP has also been reported as a beneficial agent in
reducing inflammation due to lung infection [24].
Hyperuricemic effect of copper complexes and its nanoparti-
cles have also been studied in recent years [26, 27]. From a
previous study, it has been declared that during inflammation,
the rate of oxidative stress increases. One of the studies report-
ed that CuO-NPs are also responsible for the reduction of
oxidative stress. Streptomyces pseudogriseolus Acv-11 strain
was used to prepare CuO-NPs which was responsible for the
better response in terms of scavenging and total antioxidant
activity [28]. For this purpose, we have designed a study to
illustrate the potential effects of CuO-NPs on antioxidant ac-
tivity and histopathology of MSU-induced gouty BALB/c
mice by intra-articular injection of monosodium urate crystals.
We also observed the effect of CuO-NPs in comparison with
commercially available drug allopurinol for the treatment of
gouty arthritis for the reduction of oxidative stress.
In addition to the inflammatory process in gouty arthritis,
oxidative stress (OS) is one of the important parameters associ-
ated with gout. The oxidative stress condition is triggered by the
increased production of reactive oxygen species (ROS) and cy-
tokines as they are pro-inflammatory in nature [12]. The gener-
ation of ROS is considered an essential constituent in many
physiological procedures such as the bactericidal activity of
phagocytes, signal transduction, redox state, and modulation of
the cell cycle [13]. OS is the fluctuation between ROS production
and antioxidant mechanisms [14]. As a result of its high chemical
reactivity, ROS can damage various biomolecules, such as mem-
brane lipid peroxidation, in which the oxidation of fatty acids or
lipids results in oxygen free radicals such as peroxide and DNA
damage and protein oxidative activity [15]. Another source of
ROS production in enzymatic reactions is catalyzed by
lipoxygenase, NADPH oxidase, cyclooxygenase, xanthine oxi-
dase, and autoxidation of organic compound. NADPH oxidase
from neutrophils and macrophages invades the synovial mem-
brane and serves as another significant source of superoxide
radical production [16]. This enzyme binds to the membrane
and forms a complex by using a membrane (p22phox and
gp91phox) and cytosol (p40phox, p47phox, and p67phox)-locat-
ed subunits. It has been declared that endothelial cells, fibro-
blasts, and chondrocytes are all responsible for the expression
of NADPH oxidase components and produce oxygen free radi-
cals [17]. The oxygen free radicals are responsible for damaging
endothelial cells and the extracellular matrix, resulting in the
increased migration of neutrophils to the point of inflammation.
Free oxygen radicals are converted into hydroxyl radical (HO−)
as more aggressive ROS when it interacts with free Fe+3 or Cu+2
ions [18]. Peroxidant products, particularly malonyldialdehyde
(MDA), have been identified as a secondary mediator for
hyaluronic acid depolymerization, collagen degradation, proteo-
glycan degeneration, protein oxidation, and inhibition of prolif-
erative mechanism in cartilage tissue [19].
Methods
Synthesis of CuO-NPs
CuO-NPs were synthesized by using a previously studied
chemical reduction method [29]. First, de-ionized water (120
ml) and 1.2% of starch solution were mixed with to make a
solution. A 1.2% starch solution was utilized for the prepara-
tion of 0.1 M copper sulfate solution. Ascorbic acid (0.2 M)
was prepared by using 50 ml of de-ionized water. Under vig-
orous and continuous stirring using a magnetic stirrer, in cop-
per sulfate solution, ascorbic acid was added gradually. Later
Copper is one of the important biologically essential trace
elements. At the cellular level, its function is to inhibit xan-
thine oxidase (XOD) and xanthine dehydrogenase (XDH),
which results in the reduction of purine conversion into uric
acid [20]. It has been reported from the previous studies that
the reduction or overproduction of XOD/XDH increases the

Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO...
on, de-ionized water (30 ml) was used to prepare (0.1 M)
NaOH solution which served as a reducing agent and was
added dropwise into the main solution. The temperature of
the stirring solution was kept up to 80 °C. After the addition
of NaOH, the color of the solution turned from yellow to ochre
which indicates the synthesis of copper nanoparticles. These
obtained particles were filtered and washed several times with
de-ionized water and ethanol. Finally, these particles were
dried at room temperature, and this whole process can better
be understood by Fig. 1. These copper nanoparticles were
used to make 5, 10, and 20 ppm colloidal solutions in de-
ionized water which were then orally administered to mice.
performed to investigate the crystalline structure of
CuO-NPs, while the surface morphological and elemen-
tal analysis was determined by scanning electron mi-
croscopy (SEM) and energy-dispersive X-ray spectros-
copy (EDS) using Tescan Vega 3.
Animal Model
In this experiment, BALB/c mice (25-30 g) were used
as a model organism. Thirty-six mice were obtained
from the National Institute of Health (NIH), Islamabad,
Pakistan, after which they were arbitrarily separated into
6 groups (n = 6). All the laboratory conditions were
maintained according to the standardized method before
initializing the experiment. Mice were kept in cages
under 22-25 °C room temperature with 24/7 dark-light
cycle. Mice were given diet according to previously
studied standards, which contained 6 mg/kg of copper
as an essential trace nutrient. The ingredients of the diet
are mentioned in Table 1 [30], and 0.1 mg/l of copper
was present in the water. Animal’s handling protocols
and techniques (Letter No. IIU (BI&BT)/FBAS-IBBC-
2017-4) were approved by the ethical committee of
IIUI department. The dosage scheme with the time in-
terval can be seen in Table 2.
Characterization
UV-Vis spectroscopy (Macy UV-1800PC) was utilized
for the detection of copper oxide nanoparticles, and it is
the simplest way to confirm the formation of nanopar-
ticles. A colloidal solution of copper nanoparticles was
prepared and quartz cuvette was used to detect the ab-
sorbance. The absorbance spectrum of the colloidal
sample was obtained in the range of 200–800 nm.
Structural composition of CuO-NPs was found out by
performing X-ray diffraction (XRD) using AXS D8
Advance. This parameter of characterization was
Fig. 1 Process for the synthesis of copper oxide nanoparticles [29]

Kiyani et al.
Table 1 The composition of diet given to mice [30]
Allopurinol Dosage
Ingredients
Growth diet (g/kg diet) Adult diet (g/kg diet)
Commercially available allopurinol by the brand name of Zyloric
100 mg was dissolved in de-ionized water. Group 6
hyperuricemic mice were administered with this allopurinol so-
lution at 50 mg/kg body weight dosage with the help of oral
gavage. This drug was administered once a day for a period of
3 weeks.
Cornstarch
397.486
465.692
140
155
100
40
Casein (>85% protein) 200
Dextrinized cornstarch 132
Sucrose
100
70
50
35
10
3
Soy bean oil
Fiber
50
Mineral mix
Vitamin mix
L-Cysteine
Choline bi-tartrate
35
Experimental Design
10
1.80
2.5
In order to find out the antioxidant activity and histopatholog-
ical changes of CuO-NPs on hyperuricemic BALB/c mice,
each group was provided with MSU to induce gouty arthritis,
except the control group. For the interval of 3 weeks, MSU
was injected inside the ankle joint of the mice. After that,
intraperitoneal injections of MSU were provided for the next
2 weeks. This induction of MSU caused inflammation of the
ankle joint, resulting in swelling. The diameter of the joints
was measured by a vernier caliper. Different concentrations of
CuO-NPs were prepared in distilled water by using a
sonicator. The 5, 10, and 20 ppm concentrations were synthe-
sized and administered to gouty mice by oral gavage for 3
weeks. A commercially available drug called allopurinol hav-
ing anti-hyperuricemic effect was administered to the positive
control group (50 mg/kg body weight). Previously it was seen
that these copper nanoparticles were significant in reducing
the serum uric acid levels as well as the swelling of joints in
BALB/c mice [27].
2.5
Tert-butylhydroquinone 0.014
0.008
Synthesis of Monosodium Urate Crystals
MSU crystals were synthesized by using sodium hy-
droxide (0.001 M) and 1.68 g uric acid. Both of them
were mixed at 70 °C temperature. To maintain the pH
(7.1-7.2), hydrochloric acid (HCl) and NaOH were
added. For the formation of crystals, the whole solution
was kept for 24 h at standardized room temperature.
The supernatant was discarded and the needle-shaped
crystals were washed several times using distilled water
[31].
Biochemical Analysis
Table 2 Group details and dosage given to each group
Antioxidant Enzyme Assays
Groups
Time interval
Dose scheme
Normal mice
For in vivo study, liver, muscle, and kidney tissues were col-
lected for antioxidant enzyme activity. An automatic homog-
enizer was used to make a homogenized mixture of tissue in
phosphate-buffered saline solution and later on the mixture
was centrifuged at 30,000 rpm for about half an hour. After
which, the supernatant was separated and utilized for antiox-
idant enzyme activity and total protein estimation.
Control (group 1) • 8 weeks daily
(normal feed)
• Intra-articular in- MSU-induced hyperuricemic
MSU group
(negative
control) (group
2)
jection 3 weeks
daily (once)
• Intraperitonial
injection 2 weeks
(once)
BALB/c mice
CuO-NP-I (group • 3 weeks daily
3)
Hyperuricemic orally
administered CuONPs 5
ppm
(once)
Catalase
CuO-NP-II
(group 4)
• 3 weeks daily
(once)
Hyperuricemic orally
administered CuONPs 10
ppm
Catalase (CAT) activity was performed by using the method
of Chance and Maehly [32] with some modifications.
Dilutions were prepared by utilizing a mixture of 2 ml of
phosphate buffer at pH = 7.0 and 50 ml of homogenate. The
variations in absorbance of the solution were observed at a
wavelength of 240 nm. One unit of CAT was defined as a
change in the absorbance by 0.01 unit/min.
CuO-NP-III
(group 5)
• 3 weeks daily
(once)
Hyperuricemic orally
administered CuONPs 20
ppm
Allopurinol
(group 6)
• 3 weeks daily
(once)
Hyperuricemic orally
administered allopurinol 50
mg/kg body weight

Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO...
Superoxide Dismutase
Tissue Histology
With some modifications, the method of Kakkar et al. [33]
was utilized to calculate superoxide dismutase (SOD) activity.
After the reaction occurred in 1 min, readings were noted at
560 nm wavelength from the spectrophotometer. SOD values
were presented in U/mg of protein.
Liver, kidney, and muscle tissues were preserved in formalin
solution for the time of 48 h. Different concentrations of alco-
hol and xylene were used to dehydrate the tissue content. The
tissues were fixed in paraffin wax and sections (5 mm) were
cut. Section cutting of tissues was performed on a microtome.
To observe the standard histology and morphometric changes,
the sections were stained with hematoxylin and eosin stain.
Microscopic evaluation was carried out on 4 samples from
each group for muscle, liver, and kidney tissues.
Guaiacol Peroxidase
Peroxidase (POD) activity in the homogenate of tissue was mea-
sured after some modifications of the spectrophotometric method
of Chance and Maehly [32]. A total of 0.1 ml of homogenate,
0.1 ml of guaiacol, and 2.5 ml and 0.3 ml of H2O2 of phosphate
buffer were used in this assay and 470 nm wavelength was used
to measure the absorbance. One unit of POD was defined as a
change in the absorbance of 0.01 unit/min.
Statistical Analysis
For the comparison of the data obtained from different groups,
Dunnett’s multiple comparison test was used which followed
analysis of variance (ANOVA), using the IBM SPSS (version
25) software. Values were expressed as mean standard de-
viation and were considered significant at P < 0.05.
Lipid Peroxidation by TBARS
Thiobarbituric acid reactive substances (TBARS) activity was
calculated in the tissue homogenate and the results were pre-
sented in TBARS/min/ml of plasma. Homogenate (0.1 ml),
trichlorobarbituric acid (1 ml), trichloroacetic acid (0.1 ml),
and phosphate buffer (0.29 ml) were mixed in this assay. This
whole solution was heated at 95 °C for 20 min and then again
for 10 min. Furthermore, it was shifted to an ice bath before
centrifuge (2500 rpm). The spectrophotometer was used to
measure absorbance at 535 nm wavelength [34].
Results
Characterization of CuO-NPs
From the XRD analysis, the highest peak was recorded
at 36.050° of 2θ at the x-axis shown in Fig. 2(b) in
comparison to the intensity, which shows the existences
of copper oxide nanoparticles. Debye-Scherrer equation
was utilized to calculate the size of the synthesized
nanoparticles which was estimated to be around 45
nm. UV-Vis spectroscopy analysis of copper oxide
nanoparticles showed an intense peak around 372 nm
as shown in Fig. 2(a). The absorption bands for copper
nanoparticles have been reported to be in the range of
550–600 nm [36–38].
SEM of CuO-NPs determined that the synthesized particles
were in nano ranges and were also agglomerated. Figure 3
shows that the prepared nanoparticles were round in shape.
Measurement of the diameter showed that these particles
ranged between 29 and 50 nm.
Reactive Oxygen Species
Reactive oxygen species (ROS) assay was performed to iden-
tify the tissue damage before and after treatment of hyperuri-
cemia with CuO-NPs. 5 ml each of H2O2, standards and the
homogenate was mixed with 140 ml of sodium acetate buffer
solution (pH = 4.8) in a 96-well plate, and this plate was
incubated at 37 °C (5 min). N,N′-diethyl-1,4-
phenylenediamine (DEPPD) ferrous sulfate (100 ml) and the
mixed sample were added according to ratio 1:25 in each well.
The plate was incubated again for 1 min at 37 °C. The absor-
bance was recorded with a break of 15 s for 3 min using a
microplate reader at 505 nm wavelength [35].
EDS of CuO-NPs revealed the quantitative analysis of the
sample which showed copper was present in adequate
amounts. Traces of carbon were also seen. EDS analysis can
be observed in Fig. 4.
Total Protein Estimation
Assessment of Antioxidant Profile
AMEDA laboratory diagnostic kit was utilized for the deter-
mination of total protein in the tissue. Liver, muscle, and kid-
ney tissues were obtained for the total protein estimation of
protein. The results of total protein were measured by plotting
absorbance between the standard values and sample. These
protein estimation results were expressed as mg/g of tissue.
SOD, POD, and CAT
In the diseased negative control group, the SOD, POD,
and CAT values were decreased (P ˂ 0.001) significant-
ly in comparison with the normal control as shown in

Kiyani et al.
Fig. 2 a UV-Vis spectrum of
synthesized copper oxide nano-
particles. b XRD pattern of syn-
thesized copper oxide
nanoparticles
Table 3. The CuO-NP-I-treated group observed a signif-
icant decrease (P ˂ 0.001) in SOD, POD, and CAT
values. Moreover the CuO-NP-II-treated group showed
a significant decrease (P ˂ 0.001) (P ˂ 0.05) in SOD
and POD values respectively. The CAT values of the
allopurinol-treated group showed a significant decrease
(P ˂ 0.001) in comparison to the normal control. CuO-
NP-III showed a significant increase (P ˂ 0.001) when
compared with control.
Total Protein Estimation
The total protein content of the diseased negative group was
increased very significantly in comparison with the normal
control (Table 5). The other treated groups showed no signif-
icant difference in comparison to the controls.
Histopathology
Mild mononuclear infiltration was observed in kidney tissue of
the MSU-induced gouty arthritis group. Some of the interstitial
hemorrhages and destruction of epithelial cells in proximal and
distal convoluted tubules were also observed. The CuO-NP-
treated group (5, 10, 20 ppm) resulted in the reduction of inflam-
mation in kidney tissues. Different doses depicted the reduced
loss of epithelial cells. There was significant change observed in
liver tissues of the diseased and treated groups. The histopathol-
ogy of muscle cells of the MSU and nanoparticle-treated groups
showed no inflammation and physiological changes in muscle
tissues as compared to the control. The changes in histopathology
can be seen in Fig. 5.
ROS and TBARS
ROS and TBARS values of the diseased negative con-
trol group were highly significant (P ˂ 0.001) and in-
creased as compared to the normal control. The CuO-
NP-I-treated group observed a significant decrease of P
˂ 0.01 in ROS and P ˂ 0.05 in TBARS values. The
CuO-NP-II-treated group showed a significant decrease
(P ˂ 0.05) in ROS values in comparison with the con-
trol. CuO-NP-III showed a significant decrease (P ˂
0.001) when compared with the control (Table 4).

Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO...
Fig. 3 Scanning electron
microscope (SEM) image of syn-
thesized copper nanoparticles
Fig. 4 Energy-dispersive X-ray spectroscopy pattern of synthesized copper oxide nanoparticle

Kiyani et al.
Table 3 Results of SOD, POD, and CAT activity of CuONP-
Table 5 Results of total protein estimation of CuONP-administered
administered BALB/c mice
BALB/c mice
Groups (n = 6)
SOD (U/mg protein) POD (nmol) CAT (U/mg protein)
34.41 0.8 4.04 0.4 4.33 0.3
Parameters
Group (n = 6)
Parameter
Protein estimation (mg/g of tissue)
23.81 0.4
Control
Control
MSU group
CuO-NP-I
CuO-NP-II
CuO-NP-III
Allopurinol
50.24 1.0 ***
22.57 0.5
MSU group 22.58 0.6***
CuO-NP-I 26.01 0.8***
CuO-NP-II 32.65 0.9
CuO-NP-III 38.74 0.9***
Allopurinol 32.74 0.7
1.60 0.2*** 1.90 0.1 ***
2.45 0.1*** 2.57 0.03***
24.38 0.3
3.17 0.1*
6.15 0.2*** 6.35 0.1***
2.74 0.1 3.01 0.2***
3.62 0.1
24.70 0.2
24.72 0.3
Values are expressed as mean SD
*P < 0.05
Values are expressed as mean SD
*P < 0.05
**P < 0.01
**P < 0.01
***P < 0.001
***P < 0.001
The biological system of the cell gets disturbed due to exces-
sive oxidative stress. Previous studies revealed that administra-
tion of CuO-NPs will reduce the oxidative stress due to its anti-
oxidant property [41]. In this study, gout was induced by using
xanthine oxidase inhibitor known as monosodium urate crystals.
The symptoms that appeared later were significantly increased (P
˂ 0.001) in ROS and TBARS indicating an induction of gout and
increased oxidative stress due to overproduction of oxygen rad-
icals [42]. The allopurinol-treated positive control group showed
no significant changes in SOD and POD values as it has the
ability to reduce oxidative stress by inhibiting xanthine oxidase
(XO) enzyme [43]. The high doses of CuO-NPs increase the
SOD, POD, and CAT values while causing a decrease in ROS
and TBARS values. Antioxidant activity gets increased due to
the high levels of SOD, POD, and CAT. Low levels of ROS and
TBARS in the CuO-NP-III group showed that they are important
for scavenging the free radicals and boosting the antioxidant
activity [41]. It has been reported that copper inhibits XOD ac-
tivity and suppresses the formation of uric acid effectively.
Similarly, a previous study also observed a significant decrease
in serum uric acid levels upon the oral administration of turmeric
and zinc oxide nanoparticles [44, 45]. It has been concluded from
the present experiment that when hyperuricemic or gouty arthrit-
ic mice are orally administered CuO-NPs, it will significantly
improve the levels of protein and decrease oxidative stress.
From this study, it can be concluded that in the coming future,
commercially available drug such as allopurinol can be replaced
by CuO-NP-containing drug as the prolonged use of allopurinol
has major side effects. Furthermore, CuO-NP efficacy has no
changes in histopathology of the liver, muscles, and kidney.
Discussion
CuO-NPs are reported for their antioxidant, antimicrobial, and
antifungal characteristics [39]. This study aimed to evaluate the
antioxidant activity and histopathology of orally administered
CuO-NPs in MSU-induced hyperuricemia. However, there has
been no report published regarding the antioxidant activity of
CuO-NPs in MSU-induced gouty arthritic mice. Gout was in-
duced by intra-articular administration of monosodium urate
(MSU) crystals. The induction of gout was confirmed by
conducting uric acid test as well as by measuring the diameter
of the ankle joint. The results of total protein estimation depicted
the significant (P ˂ 0.001) increase in values. This might be due
to an increased level of inflammation in the synovial joint be-
cause of MSU crystal accumulation [40].
Table 4 Results of ROS and TBARS activity of CuONP-administered
BALB/c mice
Groups (n = 6)
Parameter
ROS (U/g tissue)
TBARS (nmol/min/mg)
Control
3.31 0.3
3.59 0.3
MSU group
CuO-NP-I
CuO-NP-II
CuO-NP-III
Allopurinol
6.80 0.4 ***
2.27 0.1**
1.95 0.2*
1.66 0.3***
2.92 0.5
16.54 0.4***
2.26 0.2*
2.92 0.2
1.97 0.3***
3.19 0.2
Acknowledgements The authors are grateful to the International Islamic
University, Islamabad, Pakistan, Quaid-e-Azam University, Islamabad,
Pakistan, Riphah International University, Islamabad, and National
Institute of Health, Islamabad Pakistan for providing the animal house
facility.
Values are expressed as mean SD
*P < 0.05
**P < 0.01
***P < 0.001

Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO...
Fig. 5 Histopathological slides (40X) of mouse liver, kidney, and muscle tissues in which “a” is the normal control, “b” is the MSU group, “c” is the
CuONP-I, “d” is the CuONP-II, “e” is the CuONP-III, and “f” is the allopurinol control
protocol and their antibacterial efficacy. RSC Adv 4(8):3974–3983.
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in vivo and in vitro: highlighting the role of particle surface area and
Conflict of Interest The authors declare no competing interests.
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