Assessment of DNA damage and oxidative stress in juvenilefac.ksu.edu.sa/sites/default/files/38_3.pdf · 2019. 11. 12. · cle, and liver of fish and induced oxidative stress.7,8 Ali

R E S E A R CH A R T I C L E

Assessment of DNA damage and oxidative stress in juvenileChanna punctatus (Bloch) after exposure to multi-walledcarbon nanotubes

Daoud Ali | Fawaz A. Falodah | Bader Almutairi | Saad Alkahtani | Saud Alarifi

Department of Zoology, College of Science,

King Saud University, Riyadh, Saudi Arabia

Correspondence

Saud Alarifi, Department of Zoology, College

of Science, King Saud University, Riyadh

11451, Saudi Arabia.

Email: [email protected]

Funding information

Researchers Supporting Project number (RSP-

2019/27), Grant/Award Number: RSP-

2019/27

Abstract

Multi-walled carbon nanotubes (MWCNTs) have many applications in industry and

used as additives in polymers, catalysts, anodes in lithium-battery and drug delivery.

There is little information about MWCNTs' (210 nm) genotoxic potential on juvenile

freshwater fish Channa punctatus. Therefore, in this study, we have determined the

toxic effects of MWCNTs on freshwater fish C. punctatus by assessing toxicological

endpoints such as oxidative stress, mutagenicity, and genotoxicity after acute

MWCNTs exposure for 5 days. MWCNTs LC50-96 hours value for C. punctatus was

21.8 mg/L and on this basis three different MWCNTs concentrations were selected,

that is, sub-lethal I, II, and III, for 5-days exposure trials with C. punctatus. The level of

lipid peroxidation increased in the gills and kidney of exposed fish at sub-lethal con-

centrations II and III. Contrastingly, glutathione decreased more in the gills than in

the kidney. The activity of catalase enzymes decreased more in the gills than in the

kidney at sublethal concentrations I and II. Glutathione S-transferase decreased in gill

tissue but increased in kidney tissue following sub-lethal III exposure. Moreover, the

level of glutathione reductase decreased in both tissues. In addition, MWCNTs gen-

otoxicity was confirmed by DNA damage in lymphocytes, gills, kidney cells, and pro-

duction of micronuclei (MNi) in red blood cells of freshwater fish following sub-

lethal I, II, and III exposures. In conclusion, this study revealed that application of

micronucleus and comet assays for in vivo laboratory studies using freshwater fish

for screening the genotoxic potential of MWCNTs.

K E YWORD S

Channa punctatus, comet assay, genotoxicity, MWCNTs, oxidative stress

1 | INTRODUCTION

Due to the extensive production and application of multi-walled carbon

nanotubes (MWCNTs) have increased their entry into the water bodies,

producing unexpected hazardous effects on aquatic organisms. Murray

et al.1 reported toxic mechanisms of carbon nanotubes (CNTs) on

aquatic organisms in in vivo and in vitro tests. Previously, some studies

have investigated the toxic effects of CNTs alone, and it could give

invaluable information because as CNTs was coexist with other com-

pounds in the natural environment. Srivastava2 reported that CNTs

show high affinity to adsorb on pollutants. Thus, CNTs interact with

other environmental pollutants and it affects toxic nature of pollutants.

Cimbaluk et al.3 have reported toxicity of MWCNTs on fish species.

Ecotoxicology has been recognized as an important field of study for

assessing environmental risks of CNTs.4 Acute toxic study of environ-

mental pollutants is the most popular toxicological examinations and it

Received: 6 February 2019 Revised: 13 October 2019 Accepted: 16 October 2019

DOI: 10.1002/tox.22871

Environmental Toxicology. 2019;1–9. wileyonlinelibrary.com/journal/tox © 2019 Wiley Periodicals, Inc. 1

is regularly performed to assess the toxicity of different substances

such as chemicals and wastewater effluents.4 Studies on nano-

ecotoxicology have focused on estimating lethal concentrations, evalu-

ating sub-lethal effects on organisms, and identifying mechanisms of

toxicity of engineered nanomaterials.5 LC50 value is an important

parameter of toxicity to estimate a precise dose-response relationship

between organisms and test material.6 Several researchers have

reported that nanoparticles were bio accumulated in gills, kidney, mus-

cle, and liver of fish and induced oxidative stress.7,8 Ali et al.9 reported

that micronucleus (MN) and comet tests are two most popular and sen-

sitive techniques to detect the mutagenicity and genotoxicity of CNTs

in the environment. Al-Sabti and Metcalf,10 and Norppa and Falck,11

had reported that MN assay is widely used in biomonitring of environ-

mental pollutants. Comet assay is a sensitive technique and evaluate

DNA fragmentation.12 Thus comet and MN assays have been consid-

ered potential techniques to evaluate DNA damage produced by envi-

ronmental pollutants.9,13 In this study, we investigated MWCNTs toxic

mechanism on freshwater fish C. punctatus and established new knowl-

edge about the carbon nanotubes pollution to freshwater fish.

2 | MATERIALS AND METHODS

2.1 | Chemicals and characterization of MWCNTspowder

MWCNTs were purchased from US Research Nanomaterials, Inc.

According to the MWCNTs material safety data sheet, the main ingre-

dient of the product is carbon content (>97% wt), with an outside

diameter < 7 nm, inside diameter 2-5 nm, and an average tube size

of 0.5-2 μm.

MWCNTs size in suspension was measured by dynamic light scat-

tering (DLS) (Zeta-Sizer Malvern Model Nano-ZS). Transmission elec-

tron microscopy (TEM) was used to determine the size of MWCNTs

and to confirm the DLS results. All other chemicals were procured

from the local market.

2.2 | Fish and determination of sublethalconcentrations of MWCNTs

Juvenile C. punctatus (n = 40; 6.4 ± 2.6 cm standard length; 13.7

± 6.5 g) were obtained from a local fish farm. For acclimation purposes,

fish were held in glass tank (30 L) with aerated water for 15 days and

fed boiled eggs every day. The feeding of fish was stopped prior to start

the test. The experiment was carried out in glass aquaria (10 L) under

renewable conditions, and 12:12 (hour:hour) light/dark cycles.

The acute toxicity of MWCNTs was determined in renewable sys-

tem with change of experimental water on every alternate day. The

experimental water was oxygenated with the help of showers fixed

above the test tanks. The acute exposure techniques were done

according to American Public Health Association et al.14 The stock

solution of MWCNTs (1 mg/mL) was prepared in water.

The 10 juvenile fish were exposed to six target concentrations

(1, 5, 10, 15, 30, and 60 mg/L) of MWCNTs and the test was done in

duplicate to confirm the LC50-96 hours value of MWCNTs for juvenile

C. punctatus. We have analyzed LC50-96 hours value of MWCNTs

using probit analysis method as reported by Finney.15 LC50-96 hours

value of MWCNTs was 21.8 mg/L for C. punctatus (Figure 2, Table 1).

On the basis of LC50 value the three test MWCNTs concentrations

viz., sublethal I (1/4 LC50 = ~5.5 mg/L), sub lethal II (1/2

LC50 = ~ 11 mg/L), and sublethal III (3/4 LC50 = ~ 16.4 mg/L) were

estimated.

2.3 | Determination of test water quality

The physicochemical properties of test water were analyzed according

American Public Health Association et al.14

2.4 | Oxidative stress biomarkers

Fish (n = 15 per concentration) were exposed to sublethal con-

centrations I, II, and III for 1, 3, and 5 days. Body weight (g) and both

total and standard length (cm) were measured for each specimen. Fish

blood was collected immediately from the caudal vein.16 Blood was

used for the micronucleus (MN) and comet assays.

After sacrificing the fishes, gills (0.5 g), and kidney (0.5 g) were col-

lected in lysis buffer (5 mL of 0.1 M potassium phosphate buffer

[pH 6.5], 20% [vol/vol] glycerol, 1 mM EDTA, and 1.4 mM DTT). The

tissue was homogenized using a lysis buffer, centrifuged at 13000 rpm

for 20 minutes, and the supernatant was used for estimation of

reduced glutathione (GSH), lipid peroxidation (LPO), catalase (CAT), glu-

tathione reductase (GR), and glutathione S-transferase (GST) assays.

2.4.1 | Glutathione

GSH was evaluated in the gills and kidney tissue of fish by Beutler

et al. method17 Tissue lysate (100 μL), and double distilled water

(900 μL), were mixed to 1.5 mL of the reaction mixture (m-phosphoric

acid, EDTA, and NaCl). After 5 minutes incubation of reaction mixture

at room temperature, it was centrifuged at 4000 rpm for 15 minutes

TABLE 1 The 96 hours LC50 value of multi-walled carbon nanotubes (MWCNTs) for Channa punctatus (Bloch)

Chemical Fish Upper limit Lower limit LC50−96 hours

Multi-walled corbon nanotubes Channa punctatus 25.6 19.20 21.8 mg/L

2 ALI ET AL.

at 4�C. After centrifuge, supernatant (1 mL) was mixed with 4 mL of

phosphate solution (0.3 M disodium hydrogen phosphate) and 0.5 mL of

dithio-bis-2-nitrobenzoic acid. Intensity of the reaction product was eval-

uated at 412 nm using UV-vis spectrophotometer (Shimadzu Kyoto,

Japan). The GSH content was expressed as μmol GSH/mg protein.

2.4.2 | Lipid peroxidation

The induction of LPO in the gills and kidney tissue of fish was measured

using the method of Uchiama and Mihara.18 The tissue lysate (250 μL)

was mixed to 25 μL of 10 mM butylated hydroxytoluene, o-phosphoric

acid, and 2-thiobarbituric acid (1 mL of 0.67% of solution). The mixture

was incubated at 90�C for 60 minutes. After incubation the mixture

was cooled at room temperature and absorbance was measured at

535 nm. The rate of LPO was presented as nmol of thiobarbituric acid

reactive species (TBARS) formed per hour and gram of tissue.

2.4.3 | CAT assay

Gills and kidney were homogenized in PBS and EDTA for removal of

blood debris and then centrifuged. The supernatant was put on the

ice and unused samples were stored at −80�C. Then 200 μL from

each sample and standard were added to H2O2 working reagent

(500 μL), mixed, and incubated for 30 minutes. The reaction was

stopped by adding catalase quencher. A standard curve was prepared.

Optical density (OD) was measured at 520 nm.

2.4.4 | Glutathione reductase assay

GR activity was determined in the gills and kidney tissue of fish by

using the method of Sies et al.19 Samples and standard were mixed

with assay buffer and glutathione disulfide (GSSG) for few seconds.

NADPH was then added to start the reaction. OD was determined at

340 nm every minute for 10 minutes. Then ΔOD was calculated, and

the GR activity was calculated according to the standard curve.

2.4.5 | Glutathione S-transferase assay

GST activity was determined in the gills and kidney tissue of fish using

the method of Wilce and Parker.20 The sample was added to master

mix containing PBS, GSH, and substrate. OD was measured every

minute for 5 minutes, then, ΔOD was calculated at 340 nm. The activ-

ity was calculated according to the standard curve.

2.5 | Micronucleus assay

After exposure of MWCNTs, the production of MN in red blood cells

of fish was examined according Ali et al., (2009) method.21 The slides

were prepared by smearing one drop of blood on a clean frosted glass

slide. After dry, the slide was fixed in methanol for 10 minutes, left to

air-dry at room temperature. The slide was stained with Giemsa (6%)

in water (pH 6.9) for 15 minutes. The slide was examined under a light

microscope (Leitz Wetzlar, Germany) and 100 blood cells were coun-

ted from each slide.

The frequency of MN was calculated as follows:

MN %=Number of cells containing micronucleus

Total number of cells counted× 100

2.6 | Alkaline single cell gel electrophoresis

The DNA damage in fish was evaluated using alkaline single cell gel

electrophoresis assay as described by Ali et al.21 After, isolation of lym-

phocyte, kidney, and gill cells from control and exposed fish, the comet

assay slide was prepared. The slides were screened after staining with

ethedium bromide (75 μL) and randomly 25 cells per slide (50 cells per

concentration) were scored using an image analysis system (Komet-5.5,

Kinetic Imaging) attached to a fluorescent microscope (Leica) equipped

with appropriate filters. The parameter selected for quantification of

DNA damage was percent tail DNA (ie, % Tail DNA = 100 − % Head

DNA) as determined by the comet software.

2.7 | Statistical analysis

Data are expressed as the mean ± SE. Each experiment was done

minimum three times. One-way analysis of variance (ANOVA) was

done by using SPSS software (IBM Corporation, Armonk,

New York). P values less than .05 were considered statistically

significant.

3 | RESULTS

3.1 | Physicochemical qualities test water

The quality of experimental water was measured according to

APHA14 methods and presented in Table 2. In this experiment, the

water temperature ranged from 24.9 to 25.5�C and pH from 7.2 to

7.8. The concentration of DO (dissolved oxygen) ranged from 6.8 to

7.0 mg/L and the water conductivity was 269 μM/cm. The level of

chloride, total hardness, and total alkalinity were 46.4, 170, and

258 mg/L as CaCO3, respectively.

3.2 | Characterization of MWCNTs

MWCNTs toxicity depends on length, surface modifications, agglom-

eration state, and purity.22 Fraczek-Szczypta et al.22 have investigated

ALI ET AL. 3

the effect of physiochemical properties of MWCNTs on macrophage

RAW 264.7 cell line. In this experiment, length, width, and agglomera-

tion of MWCNTs were checked. As shown in Figure 1A,B, MWCNTs

were fibrous with varying lengths.

Analysis of MWCNTs size in TEM images showed the average

length of MWCNTs was 210 nm (Figure 1B), and shorter than the

hydrodynamic size (mean size: 0.8 μm) measured by DLS. The result

indicates that MWCNTs rapidly aggregated in test water because of

the hydration and reduction of electrostatic repulsion.23 However,

owing to the anisotropic and fibrous morphology of MWCNTs, DLS

data cannot reveal the exact size.

3.3 | Effects of MWCNTs on the oxidative stressin fish

After exposure to MWCNTs, the level of GSH in the gills and kidney

tissues of C. punctatus significantly decreased with increasing concen-

tration and exposure time (Figure 3C,D). It was important to note that

GSH decrease was stronger in the gills than in the kidney (Figure 3C,

D). MWCNT also induced lipid peroxide level in both tissues of

treated fishes as compared to control (Figure 3A,B). In the gill tissue,

catalase enzymes decreased at lower concentration but increased

slightly at a higher concentration (Figure 3E). Contrastingly, there

were little changes in catalase concentration in the kidney tissue

(Figure 3F). The glutathione S-transferase concentration increased at

sub-lethal concentration I (Figure 4A), but decreased at sub-lethal

concentration III (Figure 4A,B). Glutathione reductase decreased after

exposure to MWCNTs (Figure 4C,D).

3.4 | Induction of micronuclei

The induction of MNi in red blood cells of C. punctatus increased sig-

nificantly with increasing concentration of MWCNTs (Figure 5A,

Table 3). We have observed the effect of exposure time on the induc-

tion of micronucleus at all concentrations. The frequency of MNi

obtained in this study is shown in Table 3.

3.5 | DNA damage

After exposure of fish to different MWCNTs concentration, we

observed DNA damage in different tissues. The maximum DNA dam-

age was found in lymphocyte cells (Figure 6A) followed by gills

(Figure 6B) and kidney, which showed the minimum DNA damage

(Figure 6C). A significant effect of the duration of exposure

TABLE 2 Physiochemical characteristics of test water

Parameters Values

Temperature 22.9-24.5�C

pH 6.78-7.50

Dissolved oxygen (mg/L) 6.56-8.06

Total hardness (as CaCo3) μg/mL 257.9-293

Chloride (μg/mL) 46.02-54.0

Conductivity (μM/cm) 246.2-295

F IGURE 1 A, TEM image of multi-walled carbon nanotubes (MWCNTs). B, Size of MWCNTs [Color figure can be viewed atwileyonlinelibrary.com]

F IGURE 2 Acute toxicity of multi-walled carbon nanotubes(MWCNTs) on juvenile freshwater fish Channa punctatus. n = 3, *P < .05vs control [Color figure can be viewed at wileyonlinelibrary.com]

4 ALI ET AL.

(P < .05) was observed in fish specimens exposed to MWCNTs

(Figure 6A-I).

4 | DISCUSSION

The exposure of nanomaterials is unavoidable, as nanomaterials

become part of our routine life. Thus, eco-nanotoxicity research is

gaining more importance. Environmental pollutants have the capabil-

ity to induce oxidative damage in aquatic organisms, especially in

fishes, through generation of free radicals. In these experiments, we

determined the toxic effect of MWCNTs on the antioxidant system

and genetic material in juvenile C. punctatus. We also endorsed the

practice of antioxidants as biomarkers for nanoparticle treatment. The

exposure of animals and humans to nanosized fiber particles induced

us to do this type of experiments. This study is even more significant

because we have observed the sub-lethal effects of MWCNTs on

freshwater fish C. punctatus by assessing mutagenic and oxidative

stress risk after MWCNTs treatment.

In this study, we have taken two target tissues such as gills and

kidney as they had been found to be the main site for nanoparticle/

nanotubes deposition. Moreover, fish gills and kidney are important

organs that are more exposed to environmental pollutants (such as

MWCNTs). Aquatic water bodies act as a sink for different types of

pollutants. Thus, there are more chances for uptake of contaminants

by fishes from food sources, sediments, and suspended particulate

matter. Livingstone24 reported that the nutritional and ecological

habits of fish depend on various types of pollutants they are exposed

to. Fish is an aquatic sentinel model for evaluating pollution and oxi-

dative damage, not only through free radical generation but also

through cell response and repair mechanisms.25 Furthermore, fishes

are more susceptible to pollutants than the terrestrial organisms, as

they provide substantial data for determination of subtle effects of

oxidative damage, genotoxicity, mutagenicity, and other adverse

effects of environmental pollutants.26

Generally, nanosize materials can produce toxicity through vari-

ous mechanisms. Several nanoparticles have oxidizing capability

through the generation of free radicals or through their ability to

inhibit cell's antioxidant systems.27 In the present study, we have

detected an increase of LPO, GST, and a decline in SOD, CAT, GR,

and GSH activities in MWCNTs-exposed groups. Therefore, our

results could demonstrate the involvement of oxidative stress in

F IGURE 3 A. Level of LPO in gills tissue. B, Level of LPO in kidney tissue. C, Level of GSH in gills tissue. D, Level of GSH in kidney tissue.E, Catalase activity in gig tissue. F. Catalase activity in kidney tissue. Each value represents the mean−SE of three experiments.*P < .05 vs control[Color figure can be viewed at wileyonlinelibrary.com]

ALI ET AL. 5

F IGURE 4 A, Level of glutathion S transferase (GST) in gills tissue. B, Level of GST in in kidney tissue. C, Level of glutathione reductase(GR) in gills tissue. D, Level of glutathione reductase (GR) in kidney tissue. Each value represents the mean r−SE of three experiments.*P < .05 vscontrol [Color figure can be viewed at wileyonlinelibrary.com]

F IGURE 5 A, Induction of MNi (%) in erythrocyte cells at different concentration NINATCNTs exposure for 1,3 and-5 days. B, Controlerythrocyte cells of Channa punctatus. C, MNi in erythrocyte cells of Channa punctatus. Each value represents the mean−SE of threeexperiments.*P < .05 vs control [Color figure can be viewed at wileyonlinelibrary.com]

6 ALI ET AL.

response to MWCNTs exposure. MWCNTs' involvement in oxidative

stress has been recently shown in the fish Poecilia reticulata that had

been exposed to CNTs.28 All these recent observations strengthen

our suggestion of MWCNTs' involvement in creating oxidative stress

in fishes. Furthermore, our results showed that MWCNTs' effects

were directly proportional to the duration of exposure and dose.

Some researchers suggested that DNA damage as determined by

the alkaline single gel electrophoresis test act as a biomarker mutage-

nicity and genotoxicity in aquatic organism for example, fish.9,29 They

advised that this method should be associated with the application of

other biomarker. Thus, alkaline single gel electrophoresis test has

been extensively applied in bio monitoring and genetic toxicology30,31

including aquatic organisms32 as a potential tool for determining the

association between DNA damage and aquatic organisms' exposure

to environmental contaminants.

In this study, exposure of fish specimens to acute MWCNTs con-

centrations induced DNA damages, which were significantly higher

than those of controls, indicating MWCNTs' genotoxic effects on

aquatic organisms. The fragmentation of nuclear material may be

related to clastogenecity of the mutagen, as increased migration of

TABLE 3 Frequency of MN induced by MWCNTs in blood erythrocytes of Channa punctatus at different concentrations and exposure times

1 day 3 days 5 days

Dosage Frequency of MN (%) ± SE Frequency of MN (%) ± SE Frequency of MN (%) ± SE

Control 0.018 ± 0.0010 0.021 ± 0.001 0.025 ± 0.005

Positive control 0.066 ± 0.006* 0.52 ± 0.01* 0.761 ± 0.0120*

Sub lethal I 0.041 ± 0.01* 0.0902 ± 0.012* 0.296 ± 0.046*

Sub lethal II 0.102 ± 0.03* 0.302 ± 0.022* 0.66 ± 0.06*

Sub lethal III 0.204 ± 0.054* 0.760 ± 0.26* 1.08 ± 0.08*

Note: Data with asterisk differ significantly (*P < 0.05 vs control) between concentration within time of exposure.

F IGURE 6 DNA damage in different tissue of Channa punctatus, A. DNA tall (%) in lymphocyte cells, B, DNA tail (%) in gill tissue (C). DNA tail(%) in kidney tissue. DNA photomicrograph of different tissue, D. Control lymphocyte cells, E. Lymphocyte cells at sublethal III exposure for day5, F. Control gill cells, G. Gill cells at sublethal III exposure for day 5, H. Control kidney cells, 1. Kidney cells at sublethal III exposure for day5. Each value represents the mean−SE of three experiments.*P < .05 vs control [Color figure can be viewed at wileyonlinelibrary.com]

ALI ET AL. 7

DNA and induction of micronuclei due to the actions of environmen-

tal mutagen has been previously reported in fishes. DNA fragmenta-

tion was determined as percent tail DNA and nearly all DNA in the tail

or with very wide tail was observed more frequently at maximum

exposure concentration of MWCNTs at fifth day.

In this experiment, we have observed significant MNi induction at

higher MWCNTs concentrations than under the control, and the fre-

quency of MNi increased with increasing MWCNTs exposure duration

and concentration. Dose and time-dependent MNi were induced in

erythrocytes of fish by water pollution, as has been reported by Talapatra

and Nandy.33 Based on the time of exposure, D'Costa et al.34 suggested

that MNi were induced in zebrafish erythrocytes as corroborated by our

study. MNi induction was reported in different tissues of Tilapia rendalli,

Oreochromis niloticus, and Cyprinus carpio following treatment with

bleomycin, cyclophosphamide, 5-fluorouracil, and mitomycin C.35 The

findings of this study highlight the importance of using erythrocyte for

MN assay and emphasize its use as an early biomarker of exposure of fish

to clastogenic pollutants in the aquatic reservoirs.

In this experiment, significant results were found with regard to

oxidative stress, MNi induction, and comet percent tail DNA after

treatment with MWCNTs of different concentrations and for different

durations. These bioassays have generated novel insights on eco-

genotoxicity of MWCNTs, on fish by using blood, gills, and kidney tis-

sues because these are always being exposed to environmental

pollutants.

ACKNOWLEDGMENTS

This research work was funded by Researchers Supporting Project

number (RSP-2019/27), King Saud University, Riyadh, Saudi Arabia.

CONFLICT OF INTEREST

The authors declare that they do not have any conflicts of interest.

AUTHOR CONTRIBUTIONS

Conceptualization: D.A., F.A. F., B.A., S.Alk., and S.Ala. Data curation:

F.A.F., and D.A. Formal analysis: F.A.F., D.A., S.Alk., S.Ala.,

B.A. Funding acquisition: S.Ala. Investigation: F.A.F. and

D.A. Methodology: D.A., F.A.F., B.A., S.Alk., and S.Ala. Project adminis-

tration: Daoud Ali, S.Alk., and S.Ala. Supervision: D.A, S.Alk., and S.Ala.

Writing—original draft: D.A. Writing—review and editing: D.A., F.A.F.,

B.A., S.Alk., and S.Ala.

ORCID

Daoud Ali https://orcid.org/0000-0002-1045-4984

Saud Alarifi https://orcid.org/0000-0001-9824-5089

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How to cite this article: Ali D, Falodah FA, Almutairi B,

Alkahtani S, Alarifi S. Assessment of DNA damage and

oxidative stress in juvenile Channa punctatus (Bloch) after

exposure to multi-walled carbon nanotubes. Environmental

Toxicology. 2019;1–9. https://doi.org/10.1002/tox.22871

ALI ET AL. 9