ORIGINAL ARTICLE

Nanofabrication (2024) 9 | 1

Histopathological alteration in Zebrash:

Unravelling the Effects of Long-Term

Copper Oxide Nanoparticle Exposure

Himanshu Guptaa, Mansee Thakurb, Navami Dayalc,

Muskaan Singhd, Jigesh Mehtae, Ankit D. Ozaf,

Chander Prakashg

Abstract: Copper Oxide nanoparticles (CuO-NPs) are widely

used and they build up in the aquatic environment and can be

harmful to aquatic life. Aquatic creatures are affected by CuO

NPs, yet the consequences of these particles have not been well

explored despite contentious toxicological results. Therefore,

this study aimed to investigate the effects of chronic exposure

to CuO-NPs on adult zebrash. The study was carried out by

conducting physicochemical characterization of commercially

procured CuO-NPs using UV/Vis, ICP-OES spectroscopy, Trans-

mission Electron Microscopy (TEM), X-ray diffraction (XRD),

and Fourier Transform Infrared Spectroscopy(FTIR). Following

characterization, adult zebrash were chronically exposed to

0.5, 1, and 3 mg/l of CuO-NPs. Characterization revealed a het-

erogeneous population of nanoparticles in the size ranges of

10-50 nm. Oxidative stress indicators and functional markers

were studied in addition to tissue histology. Chronic exposure to

adult Fish at concentrations of 1 and 3 mg/l exhibited increased

levels of stress compared to lower concentrations of 0.5 mg/l.

Observations from histology showed that the extent of tissue

damage and injuries increased with the concentration of CuO-

NPs. In conclusion, chronic exposure to ≤50nm-sized CuO-NPs

exhibited toxic repercussions in adult zebrash.

Keywords: Copper oxide nanoparticles; TEM; FTIR; oxidative

stress; histopathology; adult zebrash; FET; Acridine orange.

INTRODUCTION

The increasing demand from consumers is driving a sharp increase

in interest in nanotechnology. Diffusion, resistance to electricity, con-

ductivity of electricity, strength, chemical reactivity, magnetism, and

a broad spectrum of adaptive biological activity are amidst the distinc-

tive chemical and physical attributes that distinguish these particles

from their bulk counterparts (Mani et al., 2019;Sahooli et al., 2012).

Commercial catalysts, chemical sensing instruments, therapeutic dis-

infection, and antimicrobials are just a few of the many possibilities

for these nanoparticles. Additionally, they aid in the production of mi-

croelectronics and cosmetics (Katwal et al., 2015). CuO-NPs’ strong

antibacterial and biocidal properties, which open up a variety of bio-

medical applications, have lately attracted a lot of attention (Nations et

al., 2015; Perreault et al., 2012). Copper oxide is a metal-based semi-

conductor having distinct electrical, magnetic, and optical signals.

Article history:

Received: 22-12-2023

Revised: 27-11-2024

Accepted: 28-11-2024

a Dept. of Medical Biotechnology,

MGM School of Biomedical

Sciences, MGMIHS, Kamothe,

Navi Mumbai 410209, India.

b Dept. of Medical Biotechnology,

MGM School of Biomedical

Sciences, MGMIHS, Kamothe,

Navi Mumbai 410209, India.

Corresponding author:

chander.mechengg@gmail.com

c School of Biotechnology and

Bioinformatics, D Y Patil

University, Navi Mumbai

400614, India.

d Dept. of Medical Biotechnology,

MGM School of Biomedical

Sciences, MGMIHS, Kamothe,

Navi Mumbai 410209, India.

e Ganpat University, Ganpat

Vidyanagar, Mehsana-

Gandhinagar Highway, North

Gujarat, 384012, India.

f University Centre of Research

and Development, Chandigarh

University, Mohali, Punjab,

140413, India.

Centre of Research Impact and

Outcome, Chitkara University

Institute of Engineering and

Technology, Chitkara University,

Rajpura-140401, Punjab, India.

School of Mechanical

Engineering, Lovely Professional

University, Phagwara, India.

g University Centre of Research

and Development, Chandigarh

University, Mohali, Punjab,

140413, India.

Corresponding author:

mansibiotech79@gmail.com

https://doi.org/10.37819/nanofab.9.2030

ORIGINAL ARTICLE Himanshu Gupta et al.

2 | Nanofabrication (2024) 9

Among the many uses for them are the production

of near-infrared lters, magnetic devices for stor-

age, sensors, catalysis, semiconductors, and super-

capacitors (Zhang et al., 2014; Devi et al., 2014;

Dagher et al., 2014). The biggest disadvantage of

CuO-NPs, despite their widespread usage in many

applications, is their potential for toxicity (Baek

& An, 2011). CuO-NPS may be hazardous to both

vertebrate and invertebrate cells, as well as to mam-

mals. To assess the dangers of manufactured NMs,

their ecotoxicological evaluations are crucial. The

aquatic environment is particularly susceptible to

exposure to articial NMs because it serves as both

a natural sink for pollutants and a natural route for

their migration. (Bondarenko et al., 2013). In aquat-

ic creatures, CuO-NPs can cause acute and chronic

toxicity. CuO-NPs are cytotoxic and genotoxic to

lung epithelia, skin, peripheral blood, cancer cell

lines, DNA changes and mutations, etc., accord-

ing to in vitro investigations (Akhtar et al., 2013).

It has been found that CuO-NPs cause toxicity to

the brain, liver, lungs, and kidneys (Zhang et al.,

2014). These nanoparticles can also induce oxida-

tive stress, raise toxicity in human lung epithelial

cells, and harm DNA and mitochondria. (Ruiz et

al., 2015).

Nanotoxicology research often uses zebrash as

a model since this teleost has 70% human genetic

similarity. Nonetheless, there is now disagreement

about the mechanism behind nanotoxicity, with

several studies emphasizing different components

(Devi et al., 2014). External fertilization, a high

number of spawns, translucent embryos, and quick

development are all desirable characteristics of this

organism (Dagher et al., 2014). This model has

various benets, including fast growth and optical

transparency, which allows for easy observation of

phenotypic responses at fatal, acute, chronic, and

sub-lethal toxicological endpoints.

Using mature zebrash as animal models, the

current research investigated the effects of CuO-NP

exposure in vivo. The objective of the research was

to investigate the sensitivity of CuO-NPs in the tis-

sue of adult zebrash. Oxidative stress indicators

were assessed, including total protein content (PC),

acetylcholinesterase enzyme activity (ACHE), cat-

alyze activity (CAT), peroxidation of lipids (LPO),

superoxide dismutase activity (SOD), and reactive

oxygen species (ROS). Histological staining was

performed to examine the tissue damage. Overall,

the present work gave a clear view of CuO-NPs-in-

duced tissue damage in adult zebrash.

MATERIALS AND METHODOLOGY

2.1. Characterization of commercially

purchased CuO-Nps

The

CuO-NPs

were

commercially

procured

from

Sigma

Aldrich

and

ranged

size

from

less

than

nm.

The

physical

appearance

the

nanoparti-

cle was a powdery black substance. The sample was

formulated as a solution before characterization ex-

periments. 10 ml of Distilled water and 1 ml of CuO

nanopowder

were

combined

and

the

solution

was

thoroughly mixed with a magnetic stirrer for twen-

ty minutes. An ultrasonicator was used for ten min-

utes to homogenise the mixture. The spectra were

analyzed using the UV/Vis spectrophotometer at a

scanning

range

200

900

nm.

CuO

nanopar-

ticles

were

examined

using

transmission

electron

microscopy using an FEI Tecnai G2 F20 at 200 kV

to determine their average size and shape. With the

use

Vertex

80’s

infrared

spectroscopy

equip-

ment (a main facility at IIT Mumbai), the presence

lack

functional

groups

all

was

identied.

CuK1.5405 radiation that has been nickel-lter was

used to perform XRD on powdered material utiliz-

ing

the

RIGAKU

XRD

apparatus.

Finally,

induc-

tively

coupled

plasma

atomic

emission

spectrom-

etry

(ICP-AES)

was

used

determine

the

copper

content in the CuO-NPs solution.

2.2. Zebrash embryotoxicity

The ZEBCOG-Zebrash facility, located at the Cen-

tral Research Laboratory, MGMIHS, Navi Mumbai,

was

used

maintain

adult

zebrash.

The

aquatic

facility

equipped

with

GENDANIO

automated

recirculating Zebrash housing system which was

used to acclimatize the sh, which included a tem-

perature of 28.5°C and a 10-hour light/dark cycle.

The

sh

were

fed

with

Frippak

+300

(dry

adult

feed) twice a day and once with hatched live Arte-

mia. Adult sh were bred, and eggs were collected.

For the embryotoxicity study, a solution of varying

concentrations

CuO-NPs

was

prepared

son-

icating

for

minutes.

The

zebrash

embryos

were exposed to 0.5, 1, and 3 mg/l of CuO-NPs and

monitored for 6, 12, 24, 36, 48, 60, 72, 84, 96, 108,

and

120

hours

post-fertilization

(hpf).

included

three

test

groups

and

one

control

group

with

eggs each. The experiments were performed in trip-

licate.

The

study

was

duly

approved

the

Insti-

tutional Animal Ethics Committee prior to starting

https://doi.org/10.37819/nanofab.9.2030

Histopathological alteration in Zebrash…

ORIGINAL ARTICLE

Nanofabrication (2024), 9 | 3

stopped, after which the sh were immersed in cold

water. Oxidative stress measurements and histolog-

ical examinations were performed on the test and

control groups.

2.4. Protein content

Protein content was estimated (Dagher et al., 2014).

The technique for estimating total protein content

used bovine serum albumin (BSA) as a standard.

Distilled water was used to dilute 0.1 ml of the tis-

sue homogenate to achieve a nal concentration

of 1 ml. To the mixture, an additional 5 milliliters

of caustic copper reagent were added. After thor-

oughly mixing, the mixture was allowed to sit at

room temperature for ten minutes without being

disturbed. 0.5 mL of a 1N Folin-Ciocalteu phenol

solution was subsequently added to this tube and it

was vigorously stirred. After that, the combination

was left to incubate at room temperature for a full

twenty minutes. The brightness of the blue color

was assessed at a wavelength of 620 nm using a

blank reagent that included all necessary compo-

nents except for the tissue homogenate. Typically,

the concentration of the protein is denoted in milli-

grams (mg) per tissue.

2.5. Acetylcholinesterase enzyme

activity (ACHE)

Using the Ellman et al. technique, the ACHE activ-

ity was observed (Aksnes et al., 2019). 50μl of the

sample was added to a reaction mixture containing

100μl of 10mM 5,5′-Dithiobis (2-nitrobenzoic acid)

and 50μl of 1M potassium phosphate buffer to con-

duct the test. The addition of 20 mL of a 25 mM

solution of acetylthiocholine iodide initiated the

reaction. The whole reaction was thereafter incu-

bated at a temperature of 37 °C for 10 minutes. The

yellow hue was measured using spectrophotometry

at an emission wavelength of 412 nm, with the sam-

ple substituted with a blank that contained 50 μl of

distilled water. Thiocholine hydrolysis was used to

measure AChE activity, and the ndings were ex-

pressed as units per milligram of protein.

2.6. Oxidative stress markers

2.6.1. Reactive oxygen species (ROS)

The quantity of ROS was measured using the

Beauchamp and Fridovich method (Beauchamp

the experiments. Photographs were taken at various

stages of the zebrash embryos’ development using

an EVOS FL Auto microscope equipped with a dig-

ital

camera.

Developmental

toxicity

was

assessed

using factors such as the rate of embryonic develop-

ment and the probability of both larvae and embry-

os surviving. Embryos treated with CuO-NPs also

displayed

pericardial

oedema,

deformity

similar

to the anomalies observed in zebrash embryos ex-

posed to other toxins. Cellular death was assessed

live

embryos

ascertain

whether

exposure

the test solution caused cellular apoptosis in the ze-

brash embryo. Using Acridine Orange (AO) stain-

ing

larvae

exposed

test

amounts

hpf,

cellular

apoptosis

was

examined.

The

embryos

were

removed

hours

post

fertilization,

cleaned

phosphate-buffered

saline,

placed

Eppendorf

tubes, and left to be exposed to 10 µg/mL doses of

AO for 30 minutes at room temperature. Before vi-

sualization,

the

embryos

were

repeatedly

washed

in phosphate-buffered saline. Using carboxymeth-

cellulose

(CMC)

immobilize

the

larvae,

the

EVOS

AUTO

Imaging

microscope

was

used

monitor

the

larvae

under

GFP

lter

(520nm

emission).

2.3. Adult zebrash toxicity

All studies on adult zebrash were performed fol-

lowing

the

OECD

209

and

329

guidelines.

The

MGMIHS

Institutional

Animal

Care

Committee

gave its approval to the project. The mature zebraf-

ish were split into four groups at random, consisting

of seven sh each, and were exposed to the follow-

ing treatments for thirty days in each group:

Group 1: Controls maintained in D/W.

Group 2: Water distillate containing 0.5 mg/l

CuO-NPs

Group 3: Water distillate containing 1 mg/l

CuO-NPs

Group 4: Water distillate containing 3 mg/l

CuO-NPs

The treatment and control groups were given the

appropriate amounts of fresh CuO-NPs along with

water distillate renewed every 24 hours. Each group

received the same feed throughout the study. Contin-

uous oxygenation was provided to the sh. Follow-

ing the nal day of treatment, sh were immersed

tricaine

methane

sulfonate

(200–300

mg/l)

for

about

minutes

till

the

opercular

movement

https://doi.org/10.37819/nanofab.9.2030

ORIGINAL ARTICLE Himanshu Gupta et al.

4 | Nanofabrication (2024) 9

& Fridovich, 1971). One common technique to as-

sess superoxide generation is nitroblue tetrazolium

(NBT) reduction. Ten milligrams of tissue were ho-

mogenized using one ml of Hank’s balancing salt

solution (HBSS). Each test sample received 0.5 ml

of NBT-HBSS, and it was then incubated at 37 °C

for eight hours. The test samples were then cen-

trifuged at 1,000 rpm for 10 minutes at 4 degrees

Celsius. After that, the pellet was given three wash-

es, each using 200 μl of methanol. The pellet was

then dissolved in a mixture of 1 milliliter of 2M

KOH and 1 milliliter of DMSO. After that, mea-

surements of optical density (OD) at 630 nm were

made and compared to a reference curve made us-

ing NBT. The generation of reactive oxygen spe-

cies, or ROS, is expressed as a percentage of the

control condition.

2.6.2. Lipid peroxidation (LPO)

LPO was calculated using the protocol given by

Devasagayam and Tarachand (Devasagayam &

Tarachand, 1987). This test involved a total of

0.9 ml of reactant mixture, which included 0.5 ml

of 0.15M Tris-HCl buffer (pH 7.4), 0.15 ml of 10 M

KH2PO4, 0.1 ml of tissue homogenate, and 0.25 ml

of distilled water. For 20 minutes, the tubes were

vigorously shaken in an incubator set at 37 °C. The

reaction was stopped by adding 1 ml of 10% trichlo-

roacetic acid (TCA). Thiobarbituric acid, a solution

containing 0.75 ml of 0.67% TBA, was added after

the tubes were vigorously shaken. After that, for

twenty minutes, they were submerged in water that

was boiling. We measured the color at 532 nm af-

ter the tubes had been centrifuged. Nanograms of

malondialdehyde (MDA) per milligram of protein

is the unit of expression used to describe the sample

compounds.

2.6.3. Superoxide Dismutase (SOD)

Marklund’s technique (Marklund & Marklund,

1974) was followed to assess SOD levels. The

following volumes were mixed: 0.15 ml of chlo-

roform, 0.25 ml of cold 100% ethanol, and 0.5 ml

of material extract. After stirring the mixture for

15 minutes in a mechanical shaker, it was spun

down at 13,000 rpm for another 15 minutes that fol-

lowed. Use of half a milliliter of the supernatant as

a test was performed. The auto-oxidation reaction

mixture consisted of 2 mL of Tris-HCl buffer with

a pH of 8.2, 0.5 mL of a 2-mL Pyrogallol solution,

and 2 mL of water. Initially, for three minutes, we

monitored

the

rate

pyrogallol

auto-oxidation.

Each measurement was taken at a 1-minute inter-

val. This process was thought to be fully auto-oxi-

dative. The enzyme test mixture included 2 milli-

liters of Tri-HCl buffer (pH 8.2), half a milliliter of

a 2 millimolar Pyrogallol solution, half a milliliter

of the enzyme mix, and enough water to make the

nal volume 4.5 milliliters. For the blank, 2.0 ml

of a pH 8.2 Tris-HCl buffer and 2.5 ml of distilled

water were used. After adding the enzyme, the rate

of inhibition of pyrogallol auto-oxidation was mea-

sured. Enzyme activity was measured in units per

milligram of protein.

2.6.4. Catalase activity

The method outlined (Turan, 2021) was used to cal-

culate the catalase (CAT) activity. 0.5 ml of hydro-

gen peroxide (H2O2) and 1 ml of phosphate buffer

(pH 7.0) were added to 1 ml of homogenate. To n-

ish

the

reaction,

milliliters

dichromate

acetic

acid solution were added at 0, 30, and 60 seconds.

The

mixture

was

then

placed

bath

boiling

water

for

minutes.

determine

the

protein

content,

the

absorbance

590

was

measured.

Histopathology:

After exposing the samples to CuO-NPs contin-

uously for 28 days, a histological examination was

performed. The sh were put to sleep in cold wa-

ter and then dissected to extract vital organs such

the

liver,

intestines,

brain,

pancreas,

and

mus-

cle. The organs were left to x in 10% formalin at

room temperature for a full day. The parafn wax

was used to implant the dried xed tissue. A Lei-

ca RM255 microtome was used to cut 5 μm serial

cross

sections,

which

were

subsequently

stained

with

both

eosin

and

hematoxylin

E).

The

samples were inspected using an Olympus Magnus

light

microscope

(Model

No.

11F589)

(Menke

et al.,

2011).

2.7. Statistical analysis

From

statistical

standpoint,

every

experiment

was

carried

out

triplicate.

Plotted

using

Python

3.12, packages of Pandas and Matplotlib, were used

for

analyzing

the

data.

The

data

were

shown

the

average

plus

minus

the

standard

deviation.

The

data

was

examined

for

normality

and

unity.

When p<0.05, we were said to have very signicant

differences.

https://doi.org/10.37819/nanofab.9.2030

Histopathological alteration in Zebrash…

ORIGINAL ARTICLE

Nanofabrication (2024), 9 | 5

3. RESULTS AND DISCUSSION

3.1. Characterization of commercially

purchased CuO NPs

3.1.1. UV-Vis absorption spectroscopy

Using a BioTek Epoch 2 microplate spectropho-

tometer, UV-Vis absorption was carried out at

the MGM Central Research Laboratory as shown

in Figure 1. Based on the absorption peak at

230 nm (λmax), the study veried the existence of

CuO-NPs. The spectroscopic readings were repeat-

ed for a week and fortnight, to check the stability

and to conrm the nanoparticles’ long-term endur-

ance. Over three months, it was discovered that

the nanoparticles were stable. Similar results were

achieved using a 200-600 nm range, and 398 and

527 nm were conrmed to be the peaks (A Brief

Review,” 2016).

Figure 1. UV-Vis absorption spectra of commercially purchased

CuO-NPs revealing λmax at 230nm.

3.1.2. Fourier transmission

electron microscopy

The FTIR measurements of the commercially

purchased CuO-NPs are given in Figure 2, which

was performed at the central facility of SAIFat

IIT-Mumbai using Vertex 80. FTIR analysis helped

in identifying the presence of functional groups.

Fig. 2 shows that the stretching vibrations of ali-

phatic C-H bonds are reected by the prominent

peaks at 3434, 1707, 1464, and 1229 cm–1, respec-

tively. Copper oxide nanoparticles’ Cu (I)-O vi-

brations are corresponding to the 610 and 505 cm1

peaks, respectively. The peaks and matching bonds

in O-H, =C-H, C-C bonds, etc. showed similar nd-

ings (Kadam et al., 2020).

3.1.3. Inductively coupled plasma atomic

emission spectroscopy

ICP-AES, an outsourced elemental analysis tool, was

used to nd an 87.6% Cu concentration in the NPs.

3.1.4. X-ray diffraction analysis

The commercially obtained CuO-NPs under-

went XRD analysis using RIGAKU, and the re-

sults showed that the specimen had a structure

that was crystalline since it had distinct Bragg

peaks (Figure 3). Two primary peaks were seen

in the XRD patterns at 2θ=35.6° and 2θ=38.82°,

which are attributed to the (−111) and (111) re-

flections of the CuO phase. These reflections

are comparable to those reported (Etefagh et al.,

2017). The position 2θ of the Bragg peak is gov-

erned by Bragg’s law for X-ray diffraction. The

sharp peaks suggested that CuO nanoparticles

had a fine crystal structure with high purity.

Sharp Bragg peaks (δ- function) indicate parti-

cles having macroscopic size. The Bragg peaks,

however, broaden as the particle size decreas-

es (Marulasiddeshi et al., 2022). The presence

of broad Bragg peaks is consistent with the fact

that samples consist of nanoparticles (Ahamed

et al., 2014).

https://doi.org/10.37819/nanofab.9.2030

ORIGINAL ARTICLE Himanshu Gupta et al.

6 | Nanofabrication (2024) 9

Figure 3. Commercially acquired CuO NPs’ XRD pattern showed two primary peaks,

representing the CuO phase, at 2θ=35.6° and 2θ=38.82°. The wide Bragg peaks

are also discernible, which is consistent with the samples’ nanoparticle composition.

Figure 2. Strong peaks found in the FTIR spectra of commercially obtained CuO-NPs at 3434, 1707,

1464, and 1229 cm−1, respectively. These correspond to the OH, C=C, C–O, and aliphatic C–H stretching

vibrations. The vibration of CuO-NPs is associated with Cu (I)–O at peaks of 610 and 505 cm−1.

https://doi.org/10.37819/nanofab.9.2030

Histopathological alteration in Zebrash…

ORIGINAL ARTICLE

Nanofabrication (2024), 9 | 7

3.1.5. Transmission electron microscopy

The CuO nanoparticles that were purchased com-

mercially were micrographed using a transmission

electron microscope, FEI Tecnai G2, F30 (Bin Mo-

barak et al., 2022). The micrographs revealed that

the nanoparticles exhibited polydispersity, ie. their

shape was more or less uniform. However, the par-

ticle sizes varied between 13 and 100 nm, with the

average diameter of the nanoparticles being less

than 50 nm (Figure 4). Some visible aggregation

was also found. The results of the TEM imaging of

the commercially bought CuO-NPs were in accor-

dance (Naz et al., 2019).

Figure 4. The commercially obtained CuO NPs showed a TEM micrograph of obvious aggregation.

The particles were evenly formed, polydisperse, and ranged in size from 13 to 100 nm,

with an average diameter of less than 50 nm.

3.2. Fish embryo toxicity

Zebrash embryos were used to test the effects of

CuO-NPs at concentrations of 0.5, 1, and 3 mg/L.

The CuO-NPs had a negative impact on the embryos.

Hatching rates were normal; however, at 72 hpf, mild

oedema was visible in embryos for all concentrations.

An increase in the death rates of the embryos was

observed at 108 hpf and 120 hpf as the concentration

increased. Normal development was observed in the

embryos of the control group at 24-120 hpf. Accord-

ing to the ndings of the study, CuO-NPs were un-

able to penetrate the tissues of the zebrash embryos

and larvae. However, they did cause an increase in

the mortality rate, a delay in hatching, and a drop

in the heartbeat rate as seen in Table 1 and Figure 5.

In addition, CuO-NPs were responsible for a variety

of anomalies manifesting themselves in groups that

contained smaller amounts (Aksakal & Ciltas, 2019).

The preceding gure makes it very clear that the

degree of damage and toxicity caused by Np’s in-

creases as their concentration increases, negatively

impacting the viability of larvae and embryos. This

data makes it clear that the concentration of less than

1 mg/ml may correspond to the lower limit of detec-

tion (LC 50) for copper oxide nanoparticles smaller

than 50 nm. The test group displayed bent notochord

at 120 hpf, as indicated by arrows, pericardial ede-

ma, and embryonic death at 108 and 120 hpf.

3.3. Histopathology results

After chronic exposure to CuO-NPs, various del-

eterious effects were observed in the architecture

of major organs of adult zebrash as studied using

histopathological analysis (Yang et al., 2021).

3.3.1. Liver

Figure 6 shows the histological alterations in the

liver of zebrash subjected to CuO-Np’s., hepatic

parenchyma exhibited mild to severe necrosis, ac-

companied by disorganization of the biliary chan-

nels, at concentrations of 0.5, 1, and 3mg/l.

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ORIGINAL ARTICLE Himanshu Gupta et al.

8 | Nanofabrication (2024) 9

Vacuoles of different shapes and sizes were de-

tected in the cytoplasm of hepatocytes. The admin-

istration of a 3mg/l dosage resulted in the presence

of hepatocytes with pleomorphic nuclei exhibiting

diverse sizes and levels of activity (Carvalho et al.,

2017). In addition, the necrotic hepatocytes were

characterized by larger cells, pyknotic nuclei, and

pale cytoplasm. Observations revealed hepatocytes

with pleomorphic nuclei exhibiting varying sizes

and levels of activity, including both euchromatic

and heterochromatic regions. The scale bars mea-

sure 50 micrometers.

Figure 5. Block arrows indicate the development of edema

following exposure to 1 and 3 mg/l of CuO NPs.

Physiological

effects

24hpf 48hpf 72hpf 96hpf

Control 0.5mg/l 1mg/l 3mg/l Control 0.5mg/l 1mg/l 3mg/l Control 0.5mg/l 1mg/l 3mg/l Control 0.5mg/l 1mg/l 3mg/l

% viability

100% 100% 100% 60% 100% 60% 20%

Motility

+++++++++++↓+ + ↓ ↓

Hatching

– – – – – – – – + + + ↓– – – –

Heart beat

– – – – + + + + + + ↓ ↓ + +

dead dead

Edema Formation

–––––––––––+––

+ +

Tail Deformity

––––––––––––––––

Table 1. Endpoints recorded after being exposed to the three NP concentrations.

Figure 6. Under an optical microscope with a 100X magnication, histological sections

of the liver of zebrash revealed vacuoles of different forms in addition to mild to moderate

necrosis of the hepatic parenchyma. The 3 mg/l group’s nuclei were pleomorphic.

3.3.2. Intestine

Zebrash treated with CuO-Nps showed histolog-

ical alterations in their intestines, as seen in Fig-

ure 7. This observation suggests that the sh may

have evolved a defensive mechanism against the

toxicity since no sh died after exposure. Tox-

ic chemical exposure damages the mucosa of the

intestine and inhibits cellular growth in intestinal

tissue, disrupting the physiology of the intestinal

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Nanofabrication (2024), 9 | 9

tissue as shown by histological alterations. Inam-

mation of the lamina propria is a common charac-

teristic of damage to the intestines brought on by

toxic chemicals (Win-Shwe & Fujimaki, 2011). The

inltration of leukocytes, mostly neutrophils, is a

characteristic of inammation. Pathological sec-

tions of the gut revealed the presence of additional

zebrash inammatory cells, including monocytes

and lymphocytes. Hyperplasia of goblet cells was

seen in the groups receiving 3mg/l. These results

further support the increased hyperemia by demon-

strating the presence of white blood cells in the tis-

sue due to inammation (Yang et al., 2021; Carval-

ho et al., 2017).

Figure 7. Under an optical microscope set to 100X magnication, histological slices

of the zebrash gut revealed the presence of inammatory cells, denoted by block arrows.

3.3.3. Brain

Previous studies on the neurotoxicity of nanopar-

ticles have shown that these particles may enter

the brain and induce neurodegeneration (Car-

valho et al., 2017). Prospects of nanoparticles

on brain-targeted drug delivery also evaluated

nanoparticle-induced neurotoxicity using the ze-

brash model. As evident from the images in Fig-

ure 8, there is damage to the ciliated columnar

epithelium at the higher concentrations of 1 and

3mg/l.

Figure 8. Under an optical microscope with 100X magnication, histological sections of the zebrash

brain demonstrate damage to the ciliated columnar epithelium at greater doses of 1 and 3 mg/l.

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ORIGINAL ARTICLE Himanshu Gupta et al.

10 | Nanofabrication (2024) 9

3.3.4. Pancreas

In most tissue sections, the pancreas segments of the

group receiving treatment had modest degenerative al-

terations in the cellular components of the pancreatic

islets, with many cells exhibiting pyknotic nuclei and

irregular cytoplasmic vacuolation as shown in Fig-

ure 9. Dark exocrine pancreas and lighter endocrine

pancreas are visible in the control sections in compar-

ison to the treated groups indicated by arrows.

Figure 9. Under an optical microscope with a 100X magnication, histological slices of zebrash pancreas

reveal small degenerative alterations in the pancreatic islets as well as a large number of cells with

pyknotic nuclei and irregular cytoplasmic vacuolation. Block arrows in the control areas show that the

exocrine pancreas is darker and the endocrine pancreas is lighter than in the treatment groups.

3.3.5. Muscle

The control group’s skeletal muscle had normal

skeletal muscle histoarchitecture, whereas the

groups treated with 1 mg/l CuO-NPs displayed

modest perimysial inammation and brillary de-

generation, as shown by the arrows in Figure 10.

Vacuolar degeneration accompanied by atrophy was

seen in the groups treated with 3 mg/l CuO-NPs. A

3 mg/l exposure to CuO-NPs causes muscle toxicity,

as shown by changes in the tissue architecture. Ac-

cording to related research, adult zebrash had mus-

cle degeneration as a result of persistent exposure to

CuO-NPs that produced muscle toxicity (Win-Shwe

& Fujimaki, 2011; Samim & Vaseem, 2023).

3.4. ACHE, Protein Content

and Oxidative Stress Markers

Our ndings showed that the main internal struc-

tures of zebrash were seriously harmed by CuO-

NP concentrations of 0.5,1 and 3 mg/l. This work

is among the rst to show that CuO-NPs negatively

impact zebrash’s main organs, including stress

indicators.

Analysis was done on the outcomes of oxidative

stress indicators such as ROS, SOD, LPO, and CAT,

as well as functional markers such as Acetylcholin-

esterase (ACHE), Protein content (PC), and others.

The fall in antioxidant levels was accompanied by a

notable rise in oxidants, indicating that the CuO-NP-

induced free radicals were too strong for antioxidant

enzymes to neutralize. The overall protein content

of the muscle tissue was signicantly higher in the

0.5 mg/l, 1 mg/l, and 3 mg/l groups than in the con-

trol. However, ACHE activity was signicantly lower

in CuO-NP treated groups than in control groups. The

oxidative stress markers in adult zebrash were inves-

tigated after their exposure to CuO-NPs (Figure 11).

When comparing the sh exposed to CuO-NPs to the

control, a consistent rise in LPO (lipid peroxidation)

and ROS (reactive oxygen species) was observed. The

sh treated with CuO-NPs showed a sharp decrease

in SOD (superoxide dismutase) levels and a rise in

CAT (catalyze activity). Figure 11 shows how the

indicators changed dramatically in a dose-dependent

manner, indicating that those receiving treatment

had higher levels of oxidative stress. All other results

were consistent (Mani et al., 2019), except the CAT

result, which was shown to have risen.

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ORIGINAL ARTICLE

Nanofabrication (2024), 9 | 11

Figure 11. Graphical representation of Protein content & ACHE activity, oxidative stress

markers- SOD & CAT and ROS & LPO of control and CuO-NPs treated groups Plotted

using python 3.12, packages used are pandas and matplotlib

Figure 10. Under an optical microscope with 100X magnication, histological slices of

zebrash skeletal muscle were examined. The control group displayed normal skeletal muscle

histo-architecture, while the treatment groups displayed minor perimysial inammation and

brillary degeneration, as indicated by arrows. Groups treated with 3 mg/l CuO-NPs exhibited

atrophy along with degeneration and vacuolar degeneration, as illustrated by arrows.

https://doi.org/10.37819/nanofab.9.2030

ORIGINAL ARTICLE Himanshu Gupta et al.

12 | Nanofabrication (2024) 9

4. CONCLUSION

Zebrash are now used in toxicology as a highly de-

veloped vertebrate model. This model has been wide-

ly used for toxicological evaluation of nanoparticles

for over a decade due to its superior efciency, low

cost, and ease of implementation. The concentration

of environmental contaminants in water may be de-

termined with the use of this easy to maintain model

system. The zebrash, with the help of cutting-edge

technology, may soon replace the existing mammali-

an models in the toxicity assessment of nanomaterials.

The present study used commercially purchased

CuO-NPs. Characterization using TEM, XRD,

FTIR and UV/Vis Spectroscopy revealed the nature

of these particles to be heterogenous and the aver-

age diameter to be within 50nm. These nanoparti-

cles were used to assess Fish embryo toxicity and

study the effect of chronic exposure on adult ze-

brash. The current study showed that CuO-NPs

are harmful to the major organs of adult zebrash

in addition to exhibiting embryo toxicity. The liver,

brain, and intestines all revealed signs of injury on

histopathology examination. When compared to the

control groups, both oxidative stress and function-

al indicators were found to be signicantly elevat-

ed. Furthermore, there was an increase in protein

content in the treated groups, indicating increased

oxidative stress and toxicity that was dose-depen-

dent. The majority of the documented impacts are

caused by oxidative stress, which is inuenced by

the CuO-NPs’ size, shape, and solubility. More oxi-

dative stress resulted in the generation of free radi-

cals. Free radicals were produced as a result of the

increased oxidative stress, and the body’s antioxi-

dant defenses had difculty neutralizing them. As a

result, morphological examinations revealed tissue

damage. The current ndings will help to illumi-

nate the poorly understood mechanism of muscle

poisoning caused due to CuO-NPs. This research

is also important for limiting the harm caused by

CuO-NPs to aquatic life in the environment.

Author Contributions

H. G., M. T., concept, writing-original draft prepa-

ration, investigation, resources, data curation; N.

D, M. S. writing-review and editing, methodology,

formal analysis; H. T. & M. T. conceptualization,

A. D. O & J. P. M., supervision, project adminis-

tration, all authors have read and agreed to the pub-

lished version of the manuscript.

Funding

Nil

Acknowledgments

Special

thanks

ZEBCOG-Zebrash

Laboratory,

CRL Lab, MGMSBS, MGMIHS, for providing the

infrastructure.

Conicts of Interest

The authors declare no conict of interest.

♦

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