Effects of Silver Nanoparticles on DNA Damage in Gills of the

Ruditapes Philippinarum

Wenxia Liu

and Chenge Liu

College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

lcg1110@foxmail.com

Keywords: Silver Nanoparticles, Ruditapes Philippinarum, DNA Damage.

Abstract: With the rapid development of nanotechnology, a certain number of nanoparticles will inevitably be released

into the marine ecosystem. In this work, the effects of silver nanoparticles (Ag NPs) on DNA damage of

marine bivalve Ruditapes Philippinarum were evaluated. The clams were exposed to 0, 10 and 40 μg/L of Ag

NPs for 14 days respectively, and samples are performed at the 0, 3, 7, 14 days. The results showed that that

Ag NPs were considered to cause genotoxic effect on clam gills, and induced a time-dependent increase of

DNA damage. The gills are more sensitive to high Ag NPs concentration. The genotoxicity developed in a

dose- and time-dependent manner.

1 INTRODUCTION

Silver nanoparticles (Ag NPs) are the most

commonly used due to their unique physico-chemical

properties, such as electrical and thermal

conductivity, catalytic activity, and antibacterial

activity (Chernousova, 2013; McGillicuddy, 2017).

This fast expansion will inevitably drive the release

of nanoparticles into marine ecosystems. It is

estimated that the concentration of Ag NPs in water

is in the range of ng/L, and in the mg/kg range in the

soil and sediment (Blaser, 2008). Because aquatic

organisms are in constant contact with pollutants

through swallowing, gill entry, skin absorption, etc.,

they are more susceptible to the toxic effects of

nanoparticles than terrestrial organisms (Moore,

2006). A marine mesoderm study showed that Ag

NPs induced DNA damage and oxidative damage to

Scrobicularia plana (Buffet, 2014). Ag NPs not only

have antibacterial effects, but also have ROS-derived

oxidative stress, biofilm damage and DNA damage

(Zuykov, 2011). Therefore, the toxic effects and

mechanisms of Ag NPs on aquatic organisms have

attracted more and more attention to assess their

impact on the ecological environment and human

health.

As the most important component of cells, DNA

is critical in maintaining cell homeostasis and genetic

information transmission, and impact analysis of

aquatic DNA has proven to be a very suitable method

for assessing the genotoxicity of environmental

pollutants, enabling the detection of toxic effects of

low concentrations of pollutants in a variety of

species. Comet experiments have been applied in

previous studies to study levels of DNA damage in

marine and freshwater bivalves exposed to pollutants

(Dhawan, 2009). Comet experiments, also known as

single-cell gel electrophoresis assays (SCGE), are a

fast and sensitive technique that requires only a small

number of cells to provide information on DNA

damage and repair (Collins, 2008). Single-cell

samples are fused in a low melting point agarose gel

for lysis, followed by electrophoresis under alkaline

conditions, and after the end of the electrophoresis, it

can be observed under a fluorescence microscope that

the stained intact DNA fragments can only remain in

situ due to large molecular weights and are spherical

in shape, while smaller fragments of broken DNA

migrate to the positive electrode, forming a comet

shape (Canesi, 2012).

Ruditapes Philippinarum is often used as a

sentinel species in ecotoxicological studies due to

their ability to filter large volumes of water, leading

to contaminant (Faggio, 2018). Study found bivalve

in its organization (mainly the gills and digestive

gland) accumulation of metals and other pollutants,

and that direct contact between the gills and the

external environment can cause more serious damage

(Tice, 2000). The gills can be used as the most

Liu, W. and Liu, C.

Effects of Silver Nanoparticles on DNA Damage in Gills of the Ruditapes Philippinarum.

DOI: 10.5220/0012015300003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 163-166

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

163

sensitive tissue sites for detecting genotoxicity, and

DNA damage in gill tissues can be measured by

comet experiments to be able to assess the potential

effects of Ag NPs on the genetic aspects of clams.

Therefore, the aim of this study was to expose

Ruditapes Philippinarum to different concentrations

of Ag NPs, identify the toxic effects of Ag NPs, and

assess the effect from the perspective of genotoxicity

by determining the degree of DNA damage in the

gills of clams.

2 MATERIAL AND METHODS

2.1 Preparation of Silver Nanoparticles

Ag NPs (purity>99.7%) were purchased from Sigma-

Aldrich with the particle size specified as <100 nm, a

stock solution of 50 mg/L was prepared in ultrapure

water, sonicated for 1 h and kept in constant shaking

to breakdown particles aggregates before adding to

the exposure tanks.

2.2 Laboratory Assay

The clams, Ruditapes Philippinarum were purchased

from Zhangcun Seafood Market in Qingdao and

domesticated for 3 days under laboratory conditions

(pH=8.1; Temperature=16.3±0.5 ℃; Salinity=31;

Dissolved Oxygen=8.3). The clams with no damage,

sensitive response and similar size were divided into

three groups: control and 10 μg/L or 40 μg/L of Ag

NPs. Set three parallel for each group, and each group

has a volume of 5 L seawater. During the experiment,

the clams were not fed satisfactorily and the test

water was changed every 24 h. On the 0, 3,7, 14 days

of exposure, clams (n=2) were randomly selected

from each group and dissected immediately, cut it

with scissors to make the tissue as small as possible,

add 3 mL trypsin, mix well, pipette the cell

suspension in a water bath at 37 °C to resolve the

tissue cells into a single cell suspension. Centrifuge

at 500 r·min

-1

and take the supernatant. Then

centrifuge at 1500 r·min

-1

, remove the supernatant,

and add 1 mL of PBS (0.1 mol/L) to the centrifuge

tube, a single-cell suspension can be obtained,

followed by single-cell electrophoresis experiments.

All of the above experimental steps are performed

under dark conditions to prevent UV rays from

affecting the results. Determination of clam gill cell

DNA Olive tail moment (OTM) by CASP comet

image analysis software, OTM = (Tail Center of

Gravity Position - Head Center of Gravity Position)

× tail DNA content.

2.3 Statistical Analysis

Data of DNA damage was shown as mean ± standard

of deviation. Significant differences between

exposure groups were detected using one-way

analysis of variance (ANOVA) and only P<0.05 was

accepted as significant. Then Origin 9.2 was used for

plotting.

3 TEST RESULTS AND

DISCUSSIONS

Fig. 1 shows that the control group with a normal

nucleus with a complete head and no migration of the

tail. On the 3 days, the nucleus DNA of clam gills

showed that a gradual increase in tail length and

migration in parallel with a slight decrease in head

size, indicating the beginning of DNA damage. On

the 7 days, it can be seen from these figures that more

DNA-strand breaks of clam gills, the length of the tail

gradually increased, and the fluorescence intensity of

the tail gradually increased, indicating that DNA

damage increases with the increase of dose and

duration of Ag NPs exposure. The highest doses (40

μg/L) of Ag NPs showed a statistically significant

increase in tail intensity compared with the control

group on the 14 days. The results of the genotoxic

effect of Ag NPs varies with the doses and duration

of exposure.

ICBB 2022 - International Conference on Biotechnology and Biomedicine

164

Figure 1: Fluorescence photomicrograph of in gill of clam Ruditapes philippinarum exposed to Ag NPs.

As can be seen from Figure 2, the OTM value in

the gills was elevated at the 3 days, but there was no

significant difference compared with the control

group. On the 7th day of the experiment, the OTM

value of each treatment group was significantly

increased (P<0.05), and the injury was most

significant in the treatment group with a

concentration of 40 μg/L, and the damage intensity

increased with the duration of exposed, and the

treatment group reached the maximum value on the

14th day. The above results show that the DNA

damage of clam gills is gradually more severe with

the increase doses and duration of exposed. The

genotoxicity of zebrafish (Danio rerio) exposed to

nTiO

(1 and 10 μg/L) for 14, 21 and 28 days was

assessed using RAPD-PCR technology, the genomic

stability decreased by 37% after 14 days of exposure

and increased with duration of exposed, and the

highest genotoxic effect was observed at the

maximum concentration of nTiO

(10 μg/L) after

exposure 21 days (Rocco, 2015).

The specific mechanism of genotoxicity of

nanoparticles to clams cells is unclear, but one study

suggests that one possibility is due to their small size

(1–100 nm), Ag NPs can penetrate the nucleus

through the nuclear pores, and the Ag NPs are highly

reactive and surface-charged, interacting directly

with DNA or nuclear proteins (Joubert, 2013).

Another possibility is the release of metal ions within

the cell to induce the production of excess reactive

oxygen species, and ROS reacts with DNA molecules

to cause damage to purine and pyrimidine bases, as

well as the DNA backbone, leading to DNA damage

(Rocha, 2014). This indirect damage caused by ROS

is the main pathway to DNA damage and can lead to

physiological damage, including damage to the

reproductive system, inhibition of growth, and

damage to various organelles such as lysosomals and

mitochondria (Gomes, 2013).

10 μg/L

40 μg/L

14d

Effects of Silver Nanoparticles on DNA Damage in Gills of the Ruditapes Philippinarum

165

Figure 2: Changes of DNA damage (OTM) in gill of clam Ruditapes philippinarum exposed to Ag NPs.

4 CONCLUSION

In this study, the effects of DNA damage in clams

exposed to Ag NPs were analyzed by comet

experiments, it can be concluded that Ag NPs were

considered to cause genotoxic effect on clam gills,

and induced a time-dependent increase of DNA

damage. The gills are more sensitive to high Ag NPs

concentration. The genotoxicity developed in a dose-

and time-dependent manner.

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03714

OTM

Exposure time/da

Control

AgNPs-10μg·L

−1

AgNPs-40μg·L

−1

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