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The Genotoxicity of Different Forms of Copper Oxide (NPs & MPs) in M. modiolus Mussels

Hassien M. Alnashiri, Mark G. J. Hartl, Teresa F. Fernandes

Centre of Marine Biodiversity & Biotechnology, School of Life Sciences, Heriot-Watt University

Hassien M Alnashiri: http://www.cmbb.hw.ac.uk/people/phd/hassien-alnashiri.html

Email: hmha1@hw.ac.uk , Mobile: 07887587047

@Hassien332 Hassien Alnashiri

Prof Fernandes’s Lab 1

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① INTRODUCTION

Copper oxide nanoparticles (CuO NPs) are one type of NP used in

a wide variety of applications, such as, batteries, inks, electronic

chips and heat transfer nanofluids. The growing use of CuO NPs

has given rise to worldwide concerns regarding their environmental

release, particularly to the marine environment. The toxicity of CuO

NPs on organisms and human health is poorly studied compared to

other metal oxides such as ZnO or TiO2. Hence, it is essential to

investigate CuO NP exposure and effects on key marine organisms,

such as benthic filter feeders and compare their effects with those

of CuO microparticles (MPs).Very few studies have determined the

effect of CuO NPs on mussels, and these have concentrated solely

on oxidative stress and lipid peroxidation, but have not investigated

DNA damage or cell viability. Thus, it is crucial to address CuO NP

effects on key marine organisms and compare their toxicity with

CuO microparticles (MPs) in order to have better understanding in

regard to the toxicity of these different forms.

② AIM

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The Genotoxicity of Different Forms of Copper Oxide (NPs & MPs) in M. modiolus Mussels

Hassien M. Alnashiri, Mark G. J. Hartl, Teresa F. Fernandes Centre of Marine Biodiversity & Biotechnology, School of Life Sciences, Heriot-Watt University

Heriot-Watt University

The focus of the present study was directed towards investigating

the toxicity of CuO NPs and MPs in M. modiolus mussels. Mussels

were exposed to a range of nominal concentrations of CuO particles

(5, 10, 15 and 20μgL¯¹) along with the control for 72 hours, after

which DNA damage (the Comet assay), cell viability (flow cytometry)

and oxidative stress (SOD activity assay) were used as

ecotoxicological biomarkers.

Figure 2: Transmission electron microscopy (TEM) images of different concentrations of CuO NPs in seawater media (a) 5μgL¯¹. (b) 20μgL¯¹ (scale bar: 0.5 μm).

④ METHODS

Figure 1: (a) Modiolus modiolus mussels. (b) The collection site (Orkney Islands).

Cramond island

Figure 3: Transmission electron microscopy (TEM) images of different concentrations of CuO

MPs in seawater media. (a) 5μgL¯¹. (b) 20μgL¯¹ (scale bar: 0.5 μm).

③ MATERIALS ③ MATERIALS

A

Horse mussels (Modiolus modiolus) (Fig.1a) were collected from the North West

side of Cava Island in Scapa Flow in the Orkney Islands in approximately 20m of

water depth (Fig.1b). Mussels were then kept in the aquarium in a large tank. All

tanks contained filtered and aerated seawater (salinity: 32-34; T: 14 =C ).

(a) (b)

Copper oxide NPs and MPs stock solutions were prepared separately in sea

water, sonicated for 30 min (bath sonicator). The size and shape of CuO NPs

and MPs were characterised using Transmission Electron Microscopy (TEM)

and Dynamic Light Scattering (DLS) (Figs. 2, 3 and 4).

2

(b)(a)

Figure 4: CuO particles aggregation (nm) in seawater using Dynamic Light Scattering. (a) CuO NPs. (b) CuO MPs.

DNA damage was determined using the Comet assay following a

protocol developed by Coughlan et al (2002) for clams and adapted for

mussels by Hartl et al (2010). Cell viability of the mussel haemocytes

was determined using flow cytometry. Superoxide dismutase (SOD)

activity was assessed using SOD kit assay. M. modiolus were exposed

separately to CuO NPs and MPs at concentrations ranging from 5, 10,

15 to 20 μgL¯¹ along with control, for 72 hours. Then, all mussels were

removed and the haemocytes and gill cells extracted and prepared for

further processing. MASTS 3rd – 5th Sept 2014

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• Many thanks to my supervisors (Teresa Fernandes and Mark Hartl) for

a great supervision. In addition I would like to acknowledge Paul

Cyphus, Margaret Stobie, John Kinross, Hugh Barras, Sean

McMenamy and Majed Alshaeri for their technical support.

Furthermore, many thanks to my parents, my wife, and my financial

support Jazan University and Ministry of Higher Education in Saudi

Arabia.

⑧ ACKNOWLEDGMENTS

• CuO NPs suspended in seawater were observed to be spherical

objects in shape (Fig. 2) and almost all particulate matter had linked

together resulting in agglomerates attached to each other. CuO MPs

were seen as reticular particles in shape and less aggregated than

CuO NPs (Fig. 3).

• Both forms of CuO (NPs and MPs) can cause DNA damage in both

types of cells (haemocytes and gill) for M. modiolus mussels even at

low concentrations (5μgL¯¹) in a concentration dependent manner

(Fig. 5 & 6).

• Similarly, both forms of CuO (NPs and MPs) have the potential to

decrease the cell viability in haemocytes cells which is consistent with

the comet assay results (Table1).

• SOD activity was increased in mussels exposed to both forms of CuO

(NPs and MPs) indicating increased oxidative stress, which is the likely

cause of the DNA damage (Fig. 7).

• CuO NPs were found to be more toxic to M. modiolus than CuO MPs.

⑥ CONCLUSION

(2) Cell viability

Cell viability results were consistent with the comet assay results. Both CuO

particles (NPs & MPs) affected the viability of mussels’ haemocytes illustrated

by a significant decrease of the percentage of live cells (Table 1).

Table 1: Percentages of live cells of M. modiolus mussel’ haemocytes cells exposed to both CuO

particles (NPs & MPs) measured by flow cytometry.

Figure 6: Images of the DNA damage in the DNA tail of the mussel cells caused by different

concentrations of CuO NPs, using Zeiss Axiophot microscope (mag 40x/o.75) and scored

using Comet Assay IV (Perceptive Instruments) .(a) Control. (b) 10μgL¯¹ . (c) 20μgL¯¹ .

(1) Comet assay

Results showed that there is a significantly (P<0.001; One way

ANOVA) increased DNA damage in gill cells of M. modiolus mussels

exposed to 5, 10, 15 and 20μgL¯¹ nominal concentrations of both

CuO particles (NPs & MPs) compared to the control (Fig 5 & 6).

However, NPs were significantly more toxic at high concentrations to

M. modiolus mussels than CuO MPs (Fig. 5).

⑤ RESULTS & DISCUSSION

Figure 5: An average (±SD; n=5) Percentage of DNA damage in the gill cells of M. modiolus

mussels exposed to different concentrations of both CuO particles (NPs & MPs) over 72

hours. (a) There is a significant difference compared to the control group. (ab) significant

difference between both CuO particles. (at P<0.001 (one-way ANOVA)).

(a) (b) (c)

⑤ RESULTS & DISCUSSION

The Genotoxicity of Different Forms of Copper Oxide (NPs & MPs) in M. modiolus Mussels

Hassien M. Alnashiri, Mark G. J. Hartl, Teresa F. Fernandes Centre of Marine Biodiversity & Biotechnology, School of Life Sciences, Heriot-Watt University

3

Cont 5 µgL¯¹ 10 µgL¯¹ 15 µgL¯¹ 20 µgL¯¹0

5

10

15

20

25

30

CuO NPs

CuO MPs

DNA damage in M. modiolus gill cells

% a

vera

ge in

Tai

l

Particulate CuO concentration (µg/L)

ab

ab

ab

abab

ab

a a

No of Samples Concentration 

CuO NPs CuO MPs

2 Control 97.08% 88.90%

2 5µgL-1 95.03% 88.49%

2 10µgL-1 86.07% 87.81%

2 15µgL-1 80.22% 76.93%

2 20µgL-1 79.23% 66.67%

(3) Oxidative stress

The SOD activity in the M. modiolus gill cells showed a significant increase in

activity in treatment groups compared to the control group for both CuO

particles (NPs & MPs) (Fig.7).

Cont 5 µgL¯¹ 10 µgL¯¹ 15 µgL¯¹ 20 µgL¯¹0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

CuO NPs

CuO MPs

Particulate CuO Concentration (µgL¯¹)

% I

nh

ibit

ion

SOD activity in M. modiolus mussels

aab

ab

abab

ab

ab

a

Figure 7: Average (±SD; n=5) percent inhibition of SOD activity in the gill cells of M. modiolus mussels

exposed to different concentrations of both CuO particles (NPs & MPs) over 72 hours. (a) There is a

significant difference compared to the control group. (ab) significant difference between both CuO particles.

(at P<0.001 (one-way ANOVA)). MASTS 3rd – 5th Sept 2014