Genotoxicity of Copper, Silver and Green Synthetic Gold Nanoparticles in fish (Ctenopharyngodon idella)

doi:10.21203/rs.3.rs-2632469/v1

Download PDF

Research Article

Genotoxicity of Copper, Silver and Green Synthetic Gold Nanoparticles in fish (Ctenopharyngodon idella)

https://doi.org/10.21203/rs.3.rs-2632469/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 12 Oct, 2023

Read the published version in Biological Trace Element Research →

You are reading this latest preprint version

Grass carp (Ctenopharyngodon idella) was exposed to nanoparticles of different concentrations i.e. copper oxide nanoparticles (CuO-NPs), silver nanoparticles (Ag-NPs) and green synthetic gold nano particles (Au-NPs) each for a period of 14 and 28 days. The DNA damage in the erythrocytes of Grass carp were detected through comet assay technique. The values of Total Comet Score (TCS) were noted for the exposed concentrations with a significant increasing trend (p < 0.05) and ordered as CuO-NPs > Ag-NPs > Au-NPs. The highest TCS value of the exposed erythrocytes was recorded for CuO-NPs at 40 mg L^− 1 after 14 days of exposition period. Comparatively, TCS values of erythrocytes exposed to green synthetic Au-NPs at all the concentrations and exposed time was less as compared to the Ag-NPs and CuO-NPs. The study confirmed the adverse effects of nanoparticles on the genetic material of fish cells.

CuO-NPs

Ag-NPs

Green synthetic Au-NPs

Comet Assay

DNA

Nanotechnology was offered by Richard P. Feynman a Nobel laureate in 1959 “There’s Plenty of Room at the Bottom” [1] and has become a well-known research field since last century. Several types of materials are produced by Nanotechnology at nanoscale. Nanoparticles (NPs) are categorized as widespread class of materials comprised of particulate substances that have one dimension less than 100 nm [2]. The nanomaterials level at present is the most advanced scientifically as well as in commercial applications. [3]

The cells of living organisms are typically of 10 µm across. However, even the proteins with a typical size of 5 nm can be comparable with the proportions of minutest man-made nanoparticles. This comparison of the sizes can explore the use of nanoparticles as small probes that can spy on the cellular machinery of living organisms with less interference.[4]

Thus, these nanomaterials have been used on large scale for both industrial and practical applications. However, the first paper published regarding nanoecotoxicology in 2006 [4] has increased the environmental concerns of NPs by showing the negative effects on the growth and survival of living species [5]. Nanoparticles can enter the animal body through oral and mostly through skin and respiratory routes [6]. The extensively used NPs in various sectors eventually enter aquatic water bodies through water runoff where it effects aquatic fauna particularly fish.

Nanoparticles impairs DNA repairing, arrest cell cycle, inhibit cell viability, generate reactive oxygen species (ROS), leak lactate dehydrogenase (LDH) [7] and cause chromosomal aberrations fusions in the exposed animals [8]. The cytotoxicity and genotoxicity caused by NPs in fish is the preferred animal to study due to its potential threat to human health after consumption. Moreover, NPs of different forms, sizes, and surface have shown cytotoxic effects that has increased oxidative and endoplasmic stress in fish [9] thereby; compelling the researchers to investigate the potential toxic effects of NPs in organisms particularly fish [8].

Grass carp (Ctenopharyngodon idella) is economically important fish with largest reported production in aquaculture globally. It is native to eastern Asia, and is a large herbivorous freshwater fish of family Cyprinidae. Grass carp is considered economically important in Pakistan, as it is widely cultured throughout the country [10]. Present study exposed Grass carp to oxide, non-oxide and green synthetic NPs to analyze their respective genotoxicity.

Sample Collection and Maintenance

Yearlings of fresh water grass carps (C. idella) with average body weight (20 ± 5 g) and length (12 ± 3 cm), regardless of gender were obtained from government carp hatcheries of Peshawar city of Khyber Pakhtunkhwa. The fish samples initially were treated with to 2% KMnO4 to protect from dermal infections and were kept in a separate large glass aquarium (temperature 27 ± 1.0ºC) for acclimatization purpose for a week. Fish were fed twice a day with artificial diet. The standard physiochemical characteristics of water were regularly observed and maintained.

Experimental Procedure

Synthesis of nanoparticles

Copper oxide NPs (CuO-NPs) synthesized by the recommended method [11], an oxide silver NPs (Ag-NPs) synthesized by the suggested method of Hikmah [12] whereas a non-oxide green synthetic gold NPs (Au-NPs) was synthesized by the described method of Murlai Krishna [13] that were donated by Department of Chemistry, Kohat University of Science and Technology, Kohat.

Exposure Concentration

Experimental procedure Four groups of six fish (Ctenopharyngodon idella) each were placed in separate glass aquaria.

Control group: This was the non-exposed, healthy fish group, labelled as control group (CG).
Group I: Fish in group-I were exposed to 20mg L^-1 values of CuO-NPs, Ag-NPs and green synthetic Au-NPs in separate tanks each for duration of 14 and 28 days respectively.
Group II: Similarly, fish in group-II was exposed to 30 mg L^-1of each NPs for the 14 and 28 days’ time interval respectively.
Group III: Likewise, fish in group-III was treated with 40 mg L^-1 concentration values of the three NPs for the 14 and 28 days of time interval.

Note

Fish mortality was not recorded during the experiment.

Blood Sample Collection

Blood samples were collected from the control and exposed fish after 14 and 28 days by using sterile 2 ml syringes flushed with EDTA.

Comet Assay

The blood was analyzed by Single Cell Gel electrophoresis technique (Comet assay) for DNA damage. Comet assay performed on fish blood was adopted according to the described protocol. [14].

Visualization Of Dna Comets

A fluorescent microscope was used to observe slides. Generally, hundred cells were analyzed per slide for the migration of DNA fragments of nucleus. Sections were examined at a magnification of 20X and 40X. DNA damage was observed by measuring fluorescent extension size and movement. Photographs were taken using digital camera linked to fluorescent microscope. Four classes of comet were observed including class 0 (no DNA damage), class 1 (minor damage), class 2 (reasonable or medium damage) class 3 (extensively damage) and class 4 (highest DNA damage). Total comet score (TCS) was then calculated according to the formula, TCS = 0(n) + 1(n) + 2(n) + 3(n) + 4(n), where “n” indicates number of cells in each class [15].

The increasing concern about the genotoxic effects of NPs compelled the scientists to investigate the genotoxic effects of selected NPs on Grass carp. The genotoxicity of CuO-NPs, Ag-NPs and green synthetic Au-NPs in red blood cells of Grass carp were analyzed using single cell gel electrophoresis technique (Comet assay). The values of Total Comet Score (TCS) in the exposed erythrocytes of different concentrations of Ag-NPs, CuO-NPs and green synthetic Au-NPs with their respective exposure periods are described in detail in Table 1. The TCS values for the exposed NPs group were found to be time and dose dependent (Figs. 1–12).

Significant increasing trend (P ≤ 0.05) in TCS values of erythrocytes were calculated after exposing it to Ag-NPs, green synthetic Au-NPs and CuO-NPs each against the administered concentrations groups of 20, 30 and 40 mg L^− 1after 14 days of exposure period whereas the TCS values significantly decreased for three types of NPs against each of the exposed concentrations after 28 days of exposure period as compared to the control group. The highest and the lowest total comet score was recorded for the fish erythrocytes exposed to the three NPs among the three administered doses of 20,30 and 40 mgL^− 1. The maximum comet score among the three exposed concentrations groups was recorded at 40 mg L^− 1 for Ag-NPs, CuO-NPs and green synthetic Au-NPs i.e. 118.63 ± 5.07, 137.43 ± 5.37 and 104.24 ± 7.31 respectively after 14 days of exposition period in comparison to the values of control group (34.80 ± 2.47). Nevertheless, the lowest recorded values of TCS for the three categorized exposed NPs was 109.80 ± 5.70, 115.29 ± 3.09 and 97.51 ± 3.78 respectively after the exposure period of 28 days at the administered concentration of 40 mg L^− 1 (Table 1, Fig. 1–10).

The comet assay is a sensitive genotoxic method to evaluate single cell DNA damage. In the present investigation, positive responses in contrast to the control had been observed in fish blood against the administered nanoparticles (Fig a and b). The overall observed significant differences in the present study were distinct in the red blood cells exposed to the three different concentrations of the NPs as compared to the non-exposed group (Table 2). The red blood cells were selected as the preferred cells for studying the DNA damage in fish because of its nucleated nature yet, the damage can be diagnosed in the kidneys and gills tissue of the fish. [16] Similar published data has revealed DNA damage by Ag-NPs by breaking its double strands followed by fragmentation and fusion. [17].

Table 1. Comet classes and TCS in response to CuO-NPs, Ag-NPs and Au-Nps treatment in erythrocytes of C.idella after 14 and 28days

1.Comet ( Per100 cells analyzed ) in Response to Ag-NPs treatment in erythrocytes of C. idella after 14 days

3.Comet ( Per100 cells analyzed ) in Response to CuO-NPs treatment in erythrocytes of C. idella after 14 days

5. Comet ( Per100 cells analyzed ) in response to green synthetic Au-NPs treatment in erythrocytes of C. idella after 14 days

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Control

78.93 ±1.60

11.90±1.50

05.16 ±1.49

04.10±1.13

34.52 ±1.51

Control

78.93 ±1.60

11.90 ±1.50

05.16±1.49

04.10 ±1.13

34.52 ±1.51

Control

78.93 ±1.60

11.90±1.50

05.16 ±1.49

04.10±1.13

34.52 ±1.51

59.33 ±1.47

11.17±1.06

11.83 ±1.02

13.65±1.09

4.01±1.07

91.82 ±2.04*

56.16 ±1.27

11.50 ±1.16

12.33 ±1.23

15.50 ±2.21

4.66 ±1.07

101.30 ±5.51*

60.83 ±1.47

18.81 ±1.06

09.02 ±1.32

8.51 ±1.12.

2.82 ±1.27

73.66 ±2.02

52.67 ±2.14

16.83±1.21

13.49 ±1.61

09.84±2.72

7.16±1.21

101.80 ±3.40*

50.65 ±1.74

16.83 ±1.01

15.01 ±1.24

08.02 ±1.52

9.48 ±1.11

108.83 ±5.62*

65.16 ±1.73

13.51 ±1.14

5.07 ±1.04

4.59 ±1.36

11.68 ±1.81

84.80 ±3.03*

48.16 ±2.12

17.12±2.42

12.16 ±1.73

12.37±2.24

10.17±2.76

118.63 ±5.07*

45.16 ±1.52

14.01 ±1.09

13.16 ±1.13

13.50 ±1.24

14.15 ±2.06

137.43 ±5.37*

4 40

53.16 ±1.5

17.59 ±1.42

110.47 ±1.03

9.37 ±1.24

9.41 ±1.57

104.24 ±7.31*

2.Comet ( Per100 cells analyzed ) in Response to Ag-NPs treatment in erythrocytes of C. idella after 28 days

4.Comet ( Per100 cells analyzed ) in Response to CuO-NPs treatment in erythrocytes of C. idella after 28 days

6. Comet ( Per100 cells analyzed ) in response to green synthetic Au-NPs treatment in erythrocytes of C. idella after 28 days

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Conc(mg/L)

Class 0

Class 1

Class 2

Class 3

Class 4

TCS

Control

78.98 ±1.46

11.35±1.51

05.16 ±1.49

4.01±1.47

34.80 ±2.47

Control

78.98 ±1.46

11.35 ±1.51

05.66 ±1.54

4.01 ±1.47

34.80 ±2.47

Control

78.98 ±1.46

11.35 ±1.51

05.66 ±1.54

4.01 ±1.47

34.80 ±2.47

62.66 ±1.01

11.17 ±1.06

09.65±1.02

8.56±1.02

7.65 ±1.10

86.01 ±4.03*

60.33 ±1.52

19.66 ±1.36

07.01 ±1.18

5.50 ±1.03

7.50 ±1.07

80.18 ±2.56*

66.82 ±1.57

15.67±1.06

6.05 ±1.02

4.61 ±1.09

6.84 ±1.17

68.95 ±2.72

57.33 ±1.01

12.01±1.06

07.18±1.04

6.13±1.09

10.86 ±1.31

94.67 ±2.22*

55.50 ±1.39

20.16 ±1.41

07.50 ±1.43

5.64 ±1.02

11.17 ±1.32

96.76 ±6.27*

65.16 ±1.73

13.51 ±1.14

5.07 ±1.04

4.59 ±1.36

11.68 ±1.81

84.80 ±3.03*

52.01 ±1.12

20.31±1.42

08.33±1.73

5.01±1.24

14.34 ±1.76

109.80 ±5.70*

50.83 ±1.52

19.01 ±1.02

09.33 ±1.72

5.66 ±1.64

15.16 ±1.08

115.29 ±3.09*

60.01 ±1.82

13.50 ±1.09

7.97 ±1.13

6.02 ±1.12

12.50 ±1.26

97.51 ±3.78*

Note :Three slides per treatment represented as mean ± SD. * (Relative significance to control group).

Table 2. Comparison of TCS in erythrocytes of C.idella exposed to selected Nanoparticles

Types of NPs	Concentration mg L^-1	comet ( per 100 cells analyzed )
Types of NPs	Concentration mg L^-1	14 days	28 days
CuO-NPs	Control	34.52 ±1.51	34.80 ±2.47
	20	101.30 ±5.51	80.18 ±2.56
	30	108.83 ±5.62	96.76 ±6.27
	40	137.43 ±5.37	115.29 ±3.09
Ag-NPs	Control	34.52 ±1.51	34.80 ±2.47
	20	91.82 ±2.04	86.01 ±4.03
	30	101.80 ±3.40	94.67 ±2.22
	40	118.63 ±5.07	109.80 ±5.70
Green synthetic Au-NPs	Control	34.52 ±1.51	34.80 ±2.47
	20	73.66 ±2.02	68.95 ±2.72
	30	92.14 ±2.37	84.80 ±3.03
	40	104.24 ±7.31	97.51 ±3.78

The frequency of the comet was alienated into four classes; no damage (class 0), slight damage (class 1), moderate damage (class 2), severe damage (class 3) and completely damaged (class 4) categorized in Table 1.

In the current investigation, the frequency of comet cells was found to be dose-dependent. At the low concentrations of selected nanoparticles (10–30 mg L^− 1), the erythrocytes were first slightly damaged as represented by class 1 and then became severely damage with the increasing concentration. This has been clarified that slight or moderate damaged to genetic materials is possibly regulated by the DNA repair mechanism however; the higher concentration along with prolonged exposure damage the repair mechanism categorized as class 4 (in the completely damaged cells), thus unsuccessful to repair itself and the damage remained higher at the higher concentration and exposition period of 28 days.[18] Comparable findings were observed in erythrocytes of Labeo rohita; where the increase in the frequency of comet cells was seen with the increase in the concentration of Ag-NPs [18]. Our information is consistent with reports showing the effects of silver nanospheres in a concentration dependent manner and proved to be cytotoxic as well as clastogenic towards the fish cells [19].

Significant increase TCS at 40 mgL-1 of 14 days treatment were recorded as 137.43 ± 5.37, 118.63 ± 5.07 and 104.24 ± 7.31 for CuO-NPs, Ag-NPs and green synthetic Au-NPs respectively whereas the TCS values against the same concentration decreased to 115.29 ± 3.09, 109.80 ± 5.70 and 97.51 ± 3.78 for CuO-NPs, Ag-NPs and green synthetic Au-NPs respectively after 28 days of treatment. Such similar findings were observed where the increased frequency of comet cells were recorded showing the DNA tail migration as 48.67 ± 7.51 µm after 14 days of sampled blood and then due to DNA repair mechanism decreased to 43.0 ± 3.46 µm after 28 days of sampling [20, 21].

The frequency of severely damaged cells (class 4) was highest among all the classes of all the selected nanoparticles at 40 mg L^− 1 concentration and 28 days of exposition period.

This study of highly damaged fish cells is comparable with the previous studies of titanium nanoparticles inducing genotoxicity in rainbow and trout cells. [22–24].

It was concluded that NPs induced considerable damage to the genetic material in the test fish which causes nuclear alterations in blood erythrocytes. The damage increases when the dose or time interval of the exposure increases by producing oxidative stress. These findings are mainly focused on the toxicological effects of different NPs however, the signaling cascades in response of Ag-NPs that induce the damage and the molecular repair mechanisms are still to be exposed in the future studies.

The data insinuates the adverse effects of nanoparticles on the DNA of fish erythrocytes and aquatic species which may lead to serious concerns towards human also. This knowledge aids in further research to comprehend the molecular repair mechanism and may help to design safer nanoparticles.

Conflicts of Interest

The authors declare no conflict of interest.

Compliance with ethical standards

Ethical approval for the study was taken from the Ethical Committee, University of Peshawar.

Authors contribution

Conceptualization, Data curation, AK; MKa; investigation, MK;b ;AD; FUD, AAS; Methodology, MKa; Writing—original draft, N.S.; Supervision, M.Kc.

Funding

No funding has been received.

Data availability: The data that support the findings of this study are available from the corresponding author, [Shah N], upon reasonable request.

Feynman RP (1992) There's plenty of room at the bottom [data storage]. Journal of microelectromechanical systems 1 (1):60-66
Laurent S, Forge D, Port M, Roch A, Robic C, Vander Elst L, Muller RN (2008) Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chemical reviews 108 (6):2064-2110
Murray CB, Kagan CR, Bawendi MG (2000) Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Annual review of materials science 30 (1):545-610
Taton TA (2002) Nanostructures as tailored biological probes. TRENDS in Biotechnology 20 (7):277-279
Nations S, Wages M, Cañas JE, Maul J, Theodorakis C, Cobb GP (2011) Acute effects of Fe2O3, TiO2, ZnO and CuO nanomaterials on Xenopus laevis. Chemosphere 83 (8):1053-1061
Asharani P, Wu YL, Gong Z, Valiyaveettil S (2008) Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19 (25):255102
Hussain S, Hess K, Gearhart J, Geiss K, Schlager J (2005) In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicology in vitro 19 (7):975-983
Khan MS, Jabeen F, Qureshi NA, Asghar MS, Shakeel M, Noureen A (2015) Toxicity of silver nanoparticles in fish: a critical review. J Bio Environ Sci 6 (5):211-227
Zhao X, Wang S, Wu Y, You H, Lv L (2013) Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish. Aquatic toxicology 136:49-59
Hamid A, Khan MU, Yaqoob J, Umar A, Rehman A, Javed S, Sohail A, Anwar A, Khan M, Ali A (2016) Assessment of mercury load in river Ravi, urban sewage streams of Lahore Pakistan and its impact on the oxidative stress of exposed fish. J Bio Environ Sci 8 (4):63-72
Kshirsagar JM, Shrivastava R, Adwani PS (2017) Preparation and characterization of copper oxide nanoparticles and determination of enhancement in critical heat flux. Thermal science 21 (1 Part A):233-242
Hikmah N, Idrus N, Jai J, Hadi A Synthesis and characterization of silver-copper core-shell nanoparticles using polyol method for antimicrobial agent. In: IOP Conference Series: Earth and Environmental Science, 2016. vol 1. IOP Publishing, p 012050
Murali Krishna I, Bhagavanth Reddy G, Veerabhadram G, Madhusudhan A (2016) Eco-friendly green synthesis of silver nanoparticles using Salmalia malabarica: synthesis, characterization, antimicrobial, and catalytic activity studies. Applied Nanoscience 6 (5):681-689
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell research 175 (1):184-191
Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Molecular biotechnology 26 (3):249-261. doi:10.1385/mb:26:3:249
Arslan ÖÇ, Boyacioğlu M, Parlak H, Katalay S, Karaaslan MA (2015) Assessment of micronuclei induction in peripheral blood and gill cells of some fish species from Aliağa Bay Turkey. Marine pollution bulletin 94 (1-2):48-54
AshaRani P, Low Kah Mun G, Hande MP, Valiyaveettil S (2009) Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano 3 (2):279-290
Khan MS, Qureshi NA, Jabeen F (2017) Assessment of toxicity in fresh water fish Labeo rohita treated with silver nanoparticles. Applied Nanoscience 7 (5):167-179. doi:10.1007/s13204-017-0559-x
Wise JP, Sr., Goodale BC, Wise SS, Craig GA, Pongan AF, Walter RB, Thompson WD, Ng AK, Aboueissa AM, Mitani H, Spalding MJ, Mason MD (2010) Silver nanospheres are cytotoxic and genotoxic to fish cells. Aquatic toxicology (Amsterdam, Netherlands) 97 (1):34-41. doi:10.1016/j.aquatox.2009.11.016
Karlsson HL, Di Bucchianico S, Collins AR, Dusinska M (2015) Can the comet assay be used reliably to detect nanoparticle‐induced genotoxicity? Environmental and molecular mutagenesis 56 (2):82-96
Karlsson HL (2010) The comet assay in nanotoxicology research. Analytical and bioanalytical chemistry 398 (2):651-666
Vevers WF, Jha AN (2008) Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro. Ecotoxicology 17 (5):410-420
Reeves JF, Davies SJ, Dodd NJ, Jha AN (2008) Hydroxyl radicals (OH) are associated with titanium dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 640 (1-2):113-122
Singh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM, Maffeis TG, Wright CJ, Doak SH (2009) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials 30 (23-24):3891-3914

No competing interests reported.

Download PDF

Journal Publication

published 12 Oct, 2023

Read the published version in Biological Trace Element Research →

Editorial decision: Major revision
16 Jul, 2023
Reviews received at journal
16 Jul, 2023
Reviewers agreed at journal
17 Mar, 2023
Reviews received at journal
05 Mar, 2023
Reviewers agreed at journal
03 Mar, 2023
Reviewers invited by journal
28 Feb, 2023
Editor assigned by journal
28 Feb, 2023
Submission checks completed at journal
28 Feb, 2023
First submitted to journal
27 Feb, 2023

You are reading this latest preprint version

Genotoxicity of Copper, Silver and Green Synthetic Gold Nanoparticles in fish (Ctenopharyngodon idella)

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Sample Collection and Maintenance

Experimental Procedure

Synthesis of nanoparticles

Exposure Concentration

Blood Sample Collection

Comet Assay

Visualization Of Dna Comets

Result

Discussion

Conclusion

Declarations

Conflicts of Interest

The authors declare no conflict of interest.

References

Additional Declarations

Status:

Journal Publication

Version 1