Genotoxic Effects of Oral Ingestion of Bimetallic Gold-core/Silver-shell Nanoparticles in Mice

Christian Mueller1,2

1Department of Biological Sciences, University at Albany, State University of New York, Albany, NY, USA.

2 Cancer Research Center, University at Albany, State University of New York, Rensselaer, NY, USA.

Abstract

Silver nanoparticles (AgNPs) are miniscule particles between the sizes of

1nm and 100nm. AgNPs are incorporated into various consumer goods such as

toothbrushes, clothing, and kitchen utensils; because of their known anti-microbial

properties. There is speculation concerning the long-term health effects of their

insertion in consumer products. One possible health effect is damage to genetic

material (genotoxicity). This study examines the effects of orally ingesting AgNPs

synthesized over gold (Au) core (Au/AgNPs) and the resulting genotoxicity. This

study is important due to the growing incorporation of AgNPs into consumer goods.

Mice orally ingested Au/AgNPs over a period of 7 days. Peripheral blood

samples were taken after the initial 7 days, and in 7 days intervals over subsequent

21 days. The genotoxicity of Au/AgNP treatment was measured by three

biomarkers. 7, 8-dihydro-8-oxo-guanine, which represents oxidative damage to

DNA, -H2AX γ representing DNA double strand breaks (DSBs), and micronuclei,

which represents chromosomal fragmentation.

Results showed a positive correlation between treatment with Au/AgNPs

and an increase in frequency of -H2AX foci. Treated mice displayed an inverseγ

relationship between frequencies of 8-oxo-g observed and treatment. The

micronuclei assay results showed no relationship between treatment and frequency

of micronuclei observed. In conclusion, an increase in genotoxicity was correlated to

Au/AgNP ingestion in the -H2AX foci, but not in the other two biomarkers.γ

Previous studies show increases in frequencies of each biomarker associated with

the treatment of Au/AgNPs. Future studies should be conducted in order to

determine the long term effects of Au/AgNPs using organisms with a longer lifespan

and expanding experimentation to a longer duration.

2

Introduction

In modern society technology evolves at an exponentially large rate. New

technology can be quite controversial due to the fact that the general public, and

sometimes even companies that produce these products, do not know the acute and

long term effects this new technology will have on society, the environment, and the

individual health of the consumer. One particularly controversial new technology is

the incorporation of silver nanoparticles (AgNPs) into consumer goods.

Silver has been used in medicine as early as 750 AD, where it was used to

treat diseases such as epilepsy and cholera (Edwards-Jones 2009). The reason silver

has been used in medicine is due to its natural anti-microbial properties. At the cell

membrane level, ionic silver (Ag+) has been observed to inhibit the proton motive

force, the respiratory electron chain, and to affect membrane permeability resulting

in cell death (Percival et al. 2005; Silver et al 2006; Edward-Jones 2009). Companies

have recently been incorporating silver into consumer products, in the form of

nanoparticles, in order to exploit its anti-microbial benefits. Some consumer

products that contain silver nanoparticles are sporting goods, wound dressing,

deodorant, clothing, toothpaste, shampoo, and mouthwash (Silver et al 2006).

The interest in nanoparticles (NPs) in general, is caused by their Nano scale

size and a high surface- to-volume ratio resulting in unique physical and chemical

properties that differ from their bulk counterparts, even at the same mass dose

(Chaudry et al. 2008, Allahverdiyev et al. 2011). AgNPs used in consumer products

range in size from 5 to 250 nm (PEN 2013). Another interesting feature of AgNPs is

their ability to dissociate into Ag+. This rate of dissociation can be attributed to

silvers responsiveness to the environment. Changes in pH, temperature, enzymes,

and concentrations of salt, have all been known to alter the surface chemistry of

AgNPs. Agglomeration has been observed as a prominent effect of environmental

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changes (Walczak et al 2012). In order to prevent the dissociation and

agglomeration of AgNPs, manufacturers often use a specific coating that assists in

maintaining the integrity of the nanoparticles.

Oral ingestion of AgNPs is of particular concern due to the absorption and

distribution of particles in various tissues. Tissue distributions in rodents shows

that ingested AgNPs are absorbed by gastrointestinal tract, into the systematic

blood circulation, and are distributed to most organs. The highest levels of AgNP

absorption were found in the liver in kidney, while the lowest absorption was found

in the blood and plasma (Kim et al. 2008; Loeschner et al. 2011). The effects AgNPs

have on a biological system are relatively unknown, however studies have found a

correlation between AgNP absorption and genotoxicity (Kovvuru et al 2015).

Genotoxicity refers to damage to genetic material, DNA. DNA damage induces cell

cycle arrest in order to repair the damage, or can cause premature cell death.

Eukaryotic organisms, through evolution, have developed complex DNA repair

mechanism that work to locate, signal, and repair the damages. Sometimes DNA

damage is left unchecked or the DNA repair mechanisms can incorrectly repair DNA.

If DNA damage remains unrepaired in a timely manner or faulty repair occurs, it can

result in chromosomal damage or mutations that can proliferate into diseases such

as cancer (Dobrzynska et al 2014;. Rodriguez-Rocha et al 2011).

In this study, the genotoxic effects of orally ingested AgNPs were investigated

in vivo using mice as the biological system. Genotoxicity was observed using three

biological markers: phosphorylated Histone 2AX ( -H2AX) foci, 7, 8-dihydro-8-γoxoguanine (8-oxo-g), and micronuclei. -H2AX and 8-oxo-g were determined inγ

circulating blood leukocytes and micronuclei were determined in erythrocytes.

H2AX is a histone protein that can be phosphorylated or acetylated. Histone

proteins are highly conserved proteins that are tasked with packaging and

compacting DNA (Rakiman et al 2008). H2AX also plays an important role in DNA

damage response. In response to a double strand break (DSB), the Serine-139 amino

4

acid residue is rapidly phosphorylated (Rakiman et al 2008), serving as a sensitive

marker of DSBs. The phosphorylated conformation of H2AX, is labeled as -H2AX. -γ γH2AX creates a signal cascade that recruits many different types of DNA repair

proteins (Rakiman et al 2008). One such protein is ATM, which is an important

kinase in the cell cycle checkpoints system. ATM phosphorylates cell cycle

checkpoints, which results in cell cycle arrest allowing a time for DNA repair to

occur, recruits other DNA repair enzymes to the site of DSBs and ultimately repairs

DSBs (Podhorecka et al 2010). -H2AX is also capable of chromatin remodeling andγ

recruitment of cohesins that contribute to generating recombination repair between

two sister chromatids(Podhorecka et al 2010). By examining the amount of -H2AXγ

foci generated in a cell the number of DSBs can be quantified. In order to visualize

the -H2AX foci, cells are sequentially incubated with two antibodies. The firstγ

antibodies bind to -H2AX, while the second antibody binds to the primary antibodyγ

+ -H2AX complex. The second antibody is typically fluorescent; an example isγ

fluorescein isothiocyanate (FITC)(Ramkian et al 2008). The foci observed represent

the -H2AX foci that form at the site of a DSB. γ

While -H2AX is used as a marker of DSBs, 8-oxo-g indicates oxidativeγ

damage in the DNA such as oxidative modification of guanine residue. 8-oxo-g is a

pro-mutagenic lesion that can result in a point mutation if 8-oxo-g is not removed

and the intact DNA sequence is restored by base excision repair enzymes. Cells

positive with this particular biomarker are at risk for G-C to A-T transversion

mutations that are common in somatic forms of cancer.

The last biological marker, micronucleus indicates chromosomal damage.

More specifically micronuclei represent acentric chromosome fragments, or whole

chromosome fragments that are not included in the daughter nuclei at the end of

telophase. These fragments not included in the daughter cell due to an improper

segregation with the mitotic spindle during anaphase (Fenech et al 2011). The

fragments often remain in the cytosol where they can be observed as small, intense,

black dots. In mice, Micronucleus assays can be performed by either extracting bone

5

marrow or peripheral blood (Kasamoto et al 2013). In this experiment peripheral

blood was used. The advantages of using peripheral blood rather than extracting

blood from the bone marrow is that the mice do not need to be killed, thus blood can

be extracted at multiple time points.

Materials/Methods

Mice Treatment and peripheral blood extraction

Wild type, C57BL6/7 strain, mice were treated daily over a period of 7 days

with 4mg/kg of 20 nm diameter spherical citrated-coated gold-core/silver-shell NPs

(Au/AgNPs) (NanoComposix, San Diego, CA) or vehicle control (2 mM sodium

citrate), by means of oral ingestion (Fig 1). The gold core and silver shell comprises

8% and 92% of nanoparticle mass, respectively. The Citrate coating has a high

degree of electrostatic stabilization, a low salt solubility, and is a compatible solvent

with water (Nano-Composix). Peripheral blood was extracted after time points of 7

days of treatment and 7 days (7+7) and 14 days (7+14) days after treatment

termination (Fig 1).

Days post-treatment

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Figure 1. Days treated and days post-treatment when data was collected.

This figure illustrates the days the mice were treated with Au/AgNPs and the

subsequent days that peripheral blood was extracted and analyzed using the three

biomarkers.

Micronucleus Assay

10 blood samples, one for each mouse (n=5 mice/group), were used in the

micronucleus assay. 3 ml of blood from each sample was dropped onto a pre-

labeled, cleaned glass slide. After the blood on the slides was allowed to dry, the

slides were fixed with an ice-cold (chilled at -800C) 10% methanol solution. After

being fixed with methanol solution the samples were stained with Giemsa staining

dye (Sigma-Aldrich), and washed with water.

2000 cells were counted overall for each sample. Out of the 2000 cells each

micronucleus recognized inside of an erythrocyte was recorded. Micronuclei appear

as small, intense, black spots.

Determination of -H2AX & 8 oxo-G γ

Before preparing the slides for the two biomarkers, the coverslips need to be

cleaned by incubating them in a concentrated solution of hydrochloric acid (HCl) for

2 hours and stored in Ethanol. 20 overall coverslips were used in order to

supplement 10 for -H2AX,γ and 10 for 8 oxo-g. Cover slips were coated with poly-D-

21 days

14 days

 7

days

 

1 day

 

Days of treatment

7

lysine to facilitate cell attachment onto cover slips. Approximately 150 microliters of

100 microgram/milliliter poly-D-lysine in water is added to each coverslip,

incubated for 5 min followed by aspiration of unbound poly-D-lysine. 50 to 100

microliters of peripheral blood is transferred into a 15 ml Falcon tube. 10 volumes

of Erythrocyte lysis buffer (Sigma-Aldrich) is added to the tube and mixed by

inversion. 10 volumes of PBS buffer are added to the Falcon tube. The Falcon tube is

then loaded onto a centrifuge and spun for 10 minutes at 2500 RPM. The

supernatant is separated from the precipitate and the precipitate is re-suspended in

50 microliters of PBS buffer. 50 microliters of the cell suspension is dropped onto

each poly-D-Lysine coated coverslip. The cells are then permeated with 0.5% Triton

X-100 in PBS. After permeation, 10% FBS in PBS is supplemented to the cells and

incubated for 1 hour to block unspecific binding sites. A primary antibody solution

is prepared in 10% FBS in PBS and added to coverslips and incubated for an hour at

room temperature. Coverslips are again washed in 0.1% Triton X-100 in order to

remove unbound or nonspecifically bound antibodies. FBS is added along with a

second antibody solution. This antibody solution contains FITC-labeled antibodies

against primary antibodies (Jackson Immunoresearch). The coverslips are then

washed with 0.1% Triton X-100 one last time in order to remove unbound or

unspecifically bound secondary antibodies and then mounted on DAPI:Vectashield

medium on microscopic slides.

Analysis of -H2AX and 8-oxo-g was conducted by using a fluorescentγ

microscope and two different color filters, blue (for DAPI) and green (for FITC). Cell

nuclei are visualized by DAPI staining. -H2AX foci per cell nucleus are recorded forγ

100 cells overall. If a cell has 5 or more -H2AX foci, the cell is considered to be aγ

positive cell for -H2AX foci formation. A -H2AX appears as a small concentrated,γ γ

bright green spot in cell nucleus. When determining 8-oxo-g, a cell nucleus need to

be visible on FITC filter in order for it to be considered positive for the presence of

8-oxo-g. Confirmation of cell is performed by visualization of cell nuclei under DAPI

filter. Like -H2AX, one hundred overall cells are counted. γ

8

Statistical analysis

Results were expressed as a mean standard error of the mean. Student’s t-test

was used to estimate the statistical significance between groups. P values below

0.05 were considered statistically significant.

Results

Au/AgNP characterization

NPs were characterized by the use of Dynamic Light Scattering (DLS) and

Transmission Electron Microscopy (TEM). NP size distribution was determined DLS

(Fig. 2). NP morphology, size and size distribution were determined by the provider

(NanoComposix) using TEM (Fig. 3A). The primary size of the NPs as given by the

provider is 20 nm ± 3 nm. The Shape of NPs was determined by using TEM,

identifying mostly spherical shapes (Fig. 3A)

9

Figure 2. Nanoparticle characterization by DLS.

(a) (b)

Figure 3. (a) Shows the un-agglomerated, individual sizes of Au/AgNPs, (Nano-

Composix). (b) Displays the solution of Au/AgNPs that is orally supplemented to the

mice (Nano-Composix).

In order to determine if the cause of genotoxic damage is due to the oral

ingestion of Au/AgNPs, a negative control group that received a vehicle (2mM

sodium citrate) only was utilized. Half of the mice that generated data in this

experiment were control mice, while half were treated with Au/AgNPs.

10

Effect of Au/AgNP on DNA double strand breaks

7 days 7+7 days 7+14 days0

0.20.40.60.8

11.21.41.61.8

2

-H2AX fociγ

controlAu/AgNPs

Timepoints of blood extracted

Ave

rage

Nu

mb

er o

f pos

iiti

ve C

ells

per

1

00

cel

ls

*

*Figure 4. -H2AX foci data. The average number of positive cells for the three time γpoints are shown with error bars to account for standard error, n=5 mice/group.

* Indicates P<0.05 vs. control

The general trend displayed for the -H2AX foci shows an increase in theγ

number of positive cells in treated mice at 7 days after treatment (7+7 days time

point), when compared to other time points (Fig. 4). For this time point an average

of 1.8 ± .2 positive cells existed per 100 cells. The treated mice at 7 day time point

contained an average of 1.2± 0.2 positive cells per 100, and the 7+14 day time point

contained 1.4± 0.2 positive cells per 100. As expected, the control samples’ number

11

of positive foci throughout all three trials was consistent. The 7+7 day control

samples contained 1.0 ± 0.1 Positive cells, while the 7 day and 7+14 day samples

both contained 0.8 ± 0.1 positive cells. The increase in -H2AX foci from the 7 day toγ

the 7+7 day time point shows that there is a positive correlation between Au/AgNP

ingestion and the frequency of -H2AX foci. Control mice contained 1.5 to 1.8-foldγ

lower number of -H2AX foci when compared to the treated mice, suggesting thatγ

Au/AgNP treatment caused -H2AX foci formation, which imply DSBs induction byγ

Au/AgNPs.

Effect of Au/AgNP on oxidative DNA damage

7 days 7+7 days 7+14 days0

5

10

15

20

25

30

35

Oxidative DNA damage

controlAu/AgNPs

Timepoints of blood extraction

Ave

rage

Nu

mb

er o

f Pos

itiv

e Ce

lls

per

1

00

cel

ls

Figure 5. 8-oxo-g data. The average number of positive cells for the three time points

are shown with error bars to account for standard error n=5 mice/group. *

Indicates P<0.05 vs. control

The levels of 8-oxo-g, indicative of oxidative DNA damage, are displayed in

figure 5. The trend displayed in the graph is that the amount of positive cells

observed for the control was higher in control than the treated group for all time

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points. The 7 day control samples contained an average of 25 ± 2 positive cells,

while the 7 day treated group contained an average of 20 ± 0.3 positive cells. The

7+7 day control samples contained an average of 24 ± 1.5 positive cells, and the 7+7

day treated samples contained 20.2 ±0.1 positive cells. The 7 +14 day time points

had the highest average values compared to the other two time points. The 7+ 14

day control samples contained 29 ± 1.2 positive cells. The 7+ 14 day treated samples

contained 21 ± 0.5 positive cells. Both the control and treated groups remained

relatively constant in the average number of positive cells in each time point

collected. The consistency of each trial demonstrates that the oral ingestion of

Au/AgNPs exhibited a direct relationship with a decrease in the frequency of

oxidative DNA damage.

Effect of Au/AgNP on chromosomal damage

0

1

2

3

4

5

6

7 days 7+7 days 7+14 days

Chromosomal Damage

controlAu/AgNPs

Timepoints of blood extracted

Ave

rage

nu

mb

er o

f Mic

ron

ucl

ei p

er

20

00

red

blo

od c

ells

*

Figure 6. Micronucleus assay data. The average number of positive cells for the three

time points are shown with error bars to account for standard error. * Indicates

P<0.05 vs control.

13

Data recorded from the Micronucleus assays performed is illustrated in

Figure 6. By referring to the graph it is evident that there is a significant increase in

the number of Micronuclei observed in the treated samples between time points of

7 days and 7+7 days. The average number of micronuclei observed in the treated, 7

day samples, was 2.6 ± 0.9 micronuclei. This number increased to an average of 5.4

± 0.6 micronuclei observed in the treated 7+7 day samples. The average number of

observed micronuclei decreased slightly to 5.0 ± 0.9 in the treated samples of the

7+14 time point. However this increase should be viewed with caution and

interpreted as false positive data, given that Au/AgNP treated mice displayed lower

(7 days) or similar (7+7 and 7+14 days) frequency of micronucleated erythrocytes

compared to respective untreated controls (Fig. 6). The average control values

remained relatively constant throughout the 21 day period. The 7 day and 7+ 14 day

control samples both averaged 4.6 ± 0.3 micronuclei observed. The 7+7 day control

sample contained an average of 5.4 ± 0.2 micronuclei. In order to analyze the data it

is important to consider both increases in the control and treated samples. There is

a significant increase in the average number of observed micronuclei in the treated

samples between the 7 day and 7+7 day time points (significance level not shown on

the graph). The oral ingestion of Au/AgNPs can be attributed to this increase.

However, the lack of increase in the frequency of micronucleated cells in treatment

versus control mice implies that no effect of Au/AgNP was observed on

chromosomal damage.

Discussion

In this study, the effects of orally ingesting Au/AgNPs were examined in vivo

in wild type mice. The majority of previous studies regarding AgNPs focus on the in

vitro effects of AgNPs. However a small amount of studies have been performed

concerning the in vivo effects of AgNP ingestion. Considering the low numbers of

studies there is still much to learn about the biological mechanism and effects AgNP

ingestion has in vivo.

14

The data collected in this study provides evidence that Au/AgNPs are

genotoxic. Treated groups exhibited a greater amount of DNA damage than in

control groups for as shown by an increase in -H2AX foci, indicative of DSBsγ . The

data for 7+7 day and 7+14 day time points for -H2AX, along with the 7 day timeγ

points for micronuclei, were determined statistically significant (P<0.05). However,

in contrast to -H2AX foci data, micronucleus assay data shows that the frequencyγ

of micronucleated cells, indicative of chromosomal damage, was decreased rather

than increased as a function of Au/AgNP treatment. 8-oxo-g, a marker of oxidative

DNA damage, showed a negative correlation between Au/AgNP treatment and DNA

damage and the data was determined to be statistically insignificant.

In a previous in vivo study bone marrow was extracted from control and

intravenously treated AgNP rats (Dobrzynksa et al 2014). A micronucleus assay was

performed using bone marrow polychromatic erythrocytes. Their results showed

that there was an approximately 3-fold increase in the frequency of micronuclei

between the control and treated groups 24h after a single treatment of 20nm and

200 nm AgNPs. Micronuclei remained to be significantly elevated one week after

exposure to both 20nm and 200 nm AgNPs. After 4 weeks, micronuclei persisted at

higher frequencies only in 20nm AgNP treated rats (Dobrzynska et al 2014). In

another study performed by Patlolla et al, rats orally ingested AgNPs at different

doses over a period of 5 days (Patlolla et al 2015). Micronucleus assay data shows

that AgNPs induced a dose-related increase in the frequency of micronuclei, and

significant differences between the control and treated groups (P<0.05). The oral

administration of AgNPs for 5 days to rats enhanced the ROS level at four tested

doses as compared to the negative control group (Patlolla et al 2015). The study of

Kovvuru et al, also shows a correlation between the frequency of micronuclei and

AgNP treatment (Kovvuru et al 2015). After just one day of ingestion both wild type

mice and Myh deficient mice increased the number of micronuclei by 4-fold. After 5

days of ingestion, a five fold change was observed (Kovvuru et al 2015). These

studies all linked the ingestion of AgNPs to an increase in the frequency of

micronuclei. The data generated in this study is however inconsistent with previous

15

studies. Several reasons that may be responsible for the inconsistencies are

difference of species being observed, coating of NPs, size of NPs, composition of NPs,

the route of exposure and, most importantly, the dose of AgNPs administered. In this

study the primary biological model was mice, which differs from the studies of

Dobrzynska et al and Patlolla et al, where rats were used (Dobrzynska et al 2014,

Patlolla et al 2015). Different species could have different reactions to AgNPs. The

coating of AgNPs has also been noted to have an effect on the degree of genotoxicity.

In a study done by Anderson et al, the persistence of AgNPs in rat lungs was tested

using two different coatings, PVP and CIT (Anderson et al 2015). Their results

determined that the CIT coated AgNPs persisted longer in lung tissue than PVP

coated AgNPs. The primary coating used in this study was CIT. The route of

exposure can also play a role in the persistence of AgNPs. Dobrzynska et al

intravenously injected AgNPS to rats (Dobrzynska et al 2014), while the study of

Anderson et al used an intratracheal method of exposure (Anderson et al 2015).

When different routes of exposure are used it is difficult to compare the results,

considering that one route could be more direct than another. For example

intravenous injection is a more direct form of exposure than inhalation or oral

ingestion. Composition of NPs is also a major variable. This study used bimetallic

Au/AgNPs, with are composed of gold core and silver shell. Bimetallic NPs have

rarely been studied in vivo so there is little to compare the results of this study to.

The majority of the comparable studies have used pure AgNPs, which could have

different in vivo effects than the bimetallic Au/AgNPs.

The frequency of -H2AX foci was greater in Au/AgNP treated mice than inγ

the control group mice. The 7+7 day and 7+ 14 day time points were determined to

be statistically significant (P<0.05). The most significant change was observed in the

7+7 day time point. In the study of Kovvuru et al, treated mice showed a 2.5 fold

increase in the frequency of -H2AX positive foci (Kovvuru et al 2015). Histoneγ

proteins are positively charged and associate with negatively charged DNA.

Histones might favor binding to a coating that has a negative charge such as PVP,

resulting in a greater frequency of DSBs. Both studies indicate that ingestion of

16

AgNPs induce double stranded DNA breaks which leads to a higher frequency of γ-

H2AX positive foci.

The ingestion of Au/AgNPs in this study did not show any increase in the

amount of oxidative DNA damage in the form of 8-oxo-g. In the study of Kovvuru et

al, AgNPs increased the levels of 8-oxo-g in both the wild type and Myh deficient

mice (Kovvuru et al 2015). AgNPs induced irreversible chromosomal damages in the

bone marrow. The observed permanent genome alterations were associated with

the elevated oxidative DNA damage, increased DSBs and downregulation of DNA

repair genes (Kovvuru et al 2015). In the present study, ingestion of Au/AgNPs was

associated with a decrease in levels of 8-oxo-g and the data was determined to be

statistically insignificant (P<0.05). This data however does not offer a biologically

meaningful explanation as to why DNA damaging agent Au/AgNPs reduces oxidative

DNA damage and contradicts with other published studies demonstrating that

AgNPs induce oxidative DNA damage in vitro in cultured cells and in vivo in rodent

animals.

Whether the ingestion of Au/AgNPs is gentotoxic is a complicated question

that involves many different variables. For instance the coating, size, shape, and

composition of the NPs can have different effects in vivo. In vivo studies are

important due to the fact the effects are observable on a live organism rather than a

culture or cells. The downside to conducting an in vivo study is the inconsistency in

each organism. For instance not all mice have the same biology, some could be more

or less susceptible to oxidative DNA stress or chromosomal damage.

In conclusion there is a positive relationship between the ingestion of

Au/AgNPs and genotoxic DNA damages. A common theme developed in these

studies is that the smaller the AgNP, the greater genotoxicity it exhibits. AgNPs also

elicit more chromosomal damages in the form of micronuclei, than particles made of

titanium oxide (Dobrzynska et al 2014). More studies need to be conducted in order

to determine the long term effects of AgNP ingestion. Using a biological model with a

17

longer lifespan and increasing the experimentation period to a longer duration

would determine the long term effects of AgNP ingestion and the effects it has on the

offspring of those organisms.

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