Evaluation of Cytotoxicity and Genotoxicity of Silver

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					                                                               Nanosilver Toxicity 1

Genotoxic and Cytotoxic Effects of Silver Nanoparticles on TK6 Lymphoblast Cells
                                                                             Nanosilver Toxicity 2


Engineered nanomaterials are being increasingly used as components of consumer products.
Silver nanoparticles in particular are the most used nanomaterials due to their antibiotic,
antibacterial, and antifungal effects. However, the toxic risks associated with human exposure to
nanomaterials are not completely known. In this study, the micronucleus assay was performed
using flow cytometry to assess the cytotoxicity and genotoxicity of 5 nm uncoated silver
nanoparticles in TK6 lymphoblast cells. Cells were treated with varying concentrations (10 - 24
µg/mL) of the nanoparticles for 28 hours in duplicate. Additional cells were treated with water
and 0.75 gy of ionizing radiation to serve as the negative and positive controls, respectively. The
cytotoxicity of the nanosilver, as measured by relative cell count, relative increase in cell count ,
and relative population doubling, was significantly increased over the negative control in a dose-
dependent manner. Cell death rates in the treated cells, measured by apoptosis/necrosis of cells,
were increased to 26.4% in the highest dose, compared to the negative control, which remained
at 2.4%. The percentage of micronuclei was also enhanced in a dose-dependent manner.
Nanosilver, however, was considered as genotoxic in the in vitro micronucleus assay according
to the OECD standards. The results suggest that 5 nm silver nanoparticles are cytotoxic, but not
                                                                             Nanosilver Toxicity 3


        Nanomaterials have dimensions that range from 1 nm to 100 nm. They occur both

naturally and as a result of manmade processes (Buzea et al., 2007). In recent years, companies

have rapidly increased the use of engineered nanomaterials in consumer products. Because of

their size, nanomaterials have different properties than the corresponding bulk materials

(normal/not nanoscale). These properties make using nanomaterials very desirable for companies,

as nanomaterials have been shown to be cheaper, harder, and generally more efficient in doing

what they are intended to do. Nanomaterial use in consumer products has increased 379% from

March 2006 to August 2009. ("Woodrow Wilson" 2010)

        Silver has long been used as an antibacterial, antifungal, and antiviral agent because

while being very toxic towards bacteria, it is generally much less toxic to humans (Silver et al.,

2006 ; Bosetti et al., 2002). It has been shown that silver nanoparticles are also antimicrobial,

antifungal and antiviral towards a broad spectrum of bacteria. (Kim et al., 2007; Kim et al.,

2008; Kim et al., 2009; Lu et al., 2008). For example, silver nanoparticles have been used to

reduce infections in burns when wound dressing is coated with sputtered nanoscale silver

(Agarwal et al., 2009; Madhumathi et al., 2009), and in many other applications, such as in air

fresheners, water purifiers, food storage containers, and coatings for clothing and bandages. At

present, silver nanoparticles are the most used engineered nanomaterials. ("Woodrow Wilson"


        Because there is a growing market for products that contain nanomaterials, consumer

exposure to nanomaterials is anticipated to increase. This exposure poses a potential risk,

because the properties of nanomaterials have not been tested for as long or as in depth as bulk

materials. The specific properties of nanomaterials may potentially cause adverse biological
                                                                             Nanosilver Toxicity 4

effects, such as cytotoxicity and genotoxicity, that are different from their bulk counterparts. For

example, the surface properties of some nanomaterials can lead to the formation of reactive

oxygen species that in turn can produce cellular damage, DNA adducts, and genotoxicity while

the bulk counterparts are generally inert (Carlson et al., 2008).

       The differences between bulk and nanoscale materials generate uncertainty for measuring

the genotoxic potential of nanomaterials using current toxicity assays. At present, the amount of

research relating to the toxicity and genotoxicity of nanomaterials is limited and in some cases,

inconclusive or outright contradictory. Therefore, it is very important to assure that the current

toxicity assays used to evaluate the safety of new products are adequate to detect the potential

cytotoxicity and/or genotoxicity of nanomaterials. A program at the National Center for

Toxicological Research (NCTR), in Jefferson, AR, is currently evaluating the genotoxicity of

engineered nanomaterials using several standard assays. Five nm silver nanoparticles have been

used as test materials. Previously, the Ames, Comet, and mouse lymphoma assays have been

conducted to evaluate the genotoxicity of the nanoparticles. In this study, the in vitro

micronucleus assay was used to measure the cytotoxicity and genotoxicity of silver nanoparticles

in TK6 cells. It is hypothesized that silver nanoparticles will cause both cytotoxicity and

genotoxicity in TK6 cells due to their specific surface properties.

       The in vitro micronucleus assay is a system that detects small membrane bound DNA

fragments (micronuclei) in the cytoplasm of interphase cells to measure genotoxicity. The

number of micronuclei compared to the number of intact nuclei can be scored and thus, the in

vitro micronucleus assay can provide a comprehensive evaluation of cytogenetic damage and

cytotoxicity (OECD, 2010). Cytotoxicity can also be measured through cell counts, and various
                                                                              Nanosilver Toxicity 5

statistical measurements such as relative cell count, relative increase in cell count, relative

population doublings, and relative cloning efficiency (Fellows et al. 2008).

Materials and methods


       The micronuclei assay was performed in accordance to the Organisation for Economic

Co-operation and Development (OECD) guidelines.

Chemicals and nanomaterials

       RPMI 1640 Medium, fetal bovine serum (FBS) and penicillin/streptomycin were

purchased from ATCC (Manassas, VA). The In Vitro MicroFlowTM Kit was purchased from

Litron Laboratories (Rochester, NY). Silver nanoparticles were obtained from the

Nanotechnology Center at University of Arkansas at Little Rock in 110 µL aliquots.

Characterization of silver nanoparticles

       The size of the silver nanoparticles was determined using Transmission Electron

Microscopy (JEOL Inc.). The images were collected on a field emission JEM-2100F TEM

equipped with a CCD camera. The silver nanoparticle samples were homogeneously dispersed in

water, and one drop of the suspension was deposited on the TEM grid, dried, and evacuated

before analysis. Figure 1 shows silver nanoparticles in a range of sizes from 5 nm to 15 nm, and

particle agglomeration occurs with different sizes up to 30 nm.

       In a biological system, the properties of the nanoparticles may change due to protein

binding. The Dynamic Light Scattering technique could be used in charactering the behavior of

nanoparticles in native biological environment (i.e. medium). Therefore, the nanostructure size

and zeta potential were measured using Zetasizer (Malvern Inc.). The silver nanoparticles were
                                                                              Nanosilver Toxicity 6

suspended in deionized water, phosphate buffered saline, or medium for particle size and zeta

potential analysis. The size and surface charge characteristics of silver nanoparticles are

summarized in Table 1. The mean size of agglomerated nanosilver particles ranged from 61.2 nm

(in water) to 1608.7 nm (in medium), and the surface charge ranged from -9.37 mV to -8.20 mV.

The micrographs show a distribution of various nanoparticle sizes.

TK6 cell culture

       TK6 lymphoblast cells from ATCC (Manassas, VA) were maintained in RPMI-1640

Medium supplemented with 10% FBS and incubated at 37o C in a 5% CO2 air atmosphere cell

incubator. Cells were counted, and diluted to a concentration of 6x104 cells/mL cell medium.

Cells were then transferred to T-25 flasks in 5 mL volume, giving a total of 3x105 cells in each

flask, and allowed to grow for 24 hours. Cells were counted before treatment for cytotoxicity

analysis, as well as to ensure the cells were growing normally.


       The cells were treated with 10, 14, 16, 18, 20, and 24 µg/mL silver nanoparticles one day

after cells were transferred into flasks. Cells serving as the positive control were treated with a

radiation dose of .75 gy. Fifty µL sterilized water was added to the negative controls (The same

volume of water that the nanomaterials were suspended in). All cell cultures were prepared in

duplicates. Cells were allowed to grow in cell medium with the nanomaterials for ~28 hours, to

allow approx. 1.5-2.0 cell divisions in the vehicle (negative) control.

Flow Cytometry

       Cells were prepared for flow cytometry following the High Content Protocol 1 from the

instruction manual for the In Vitro MicroFlowTM Kit. After the 28 hour treatment perioid, treated

cells were counted using the Z1 Particle Counter from Beckman Coulter (Brea, CA) and
                                                                            Nanosilver Toxicity 7

calculations were made to find the volume of cell medium that provides 5x105 cells for the

negative control cultures. This volume was harvested from each cell culture and placed into 15

mL centrifuge tubes. Cells were then stained with multiple dyes following the kit instruction

manual to allow the flow cytometer to distinguish micronuclei. Cells were analyzed using the

FACSCanto II flow cytometer from Becton Dickinson (San Jose, CA). The stopping gate was set

at 10,000 healthy nuclei and threshold parameters were set following the instruction manual.

Cytotoxicity Analysis

       Analysis of cytotoxicity was done from calculations that used counts from the Z1 Particle

Counter as well as information gathered from the flow cytometer. Cells were counted at the

beginning of treatment and at cell harvesting. Relative cell count (RCC), relative increase in cell

count (RICC), relative population doublings (RPD), and relative nuclei to bead ratio (RNBR)

were calculated and expressed as a percentage relative to the negative controls. Cell death

percentages were also gathered from the flow cytometer.

Statistical Analysis

       Statistical analysis was performed using SigmaStat 3.11 (San Jose, CA). Linear

regression was applied to all cytotoxicity measurements to create a linear trend analysis.

Genotoxicity was measured by the micronuclei percentage in the treated samples compared to

the negative control samples. A sample with at least 2x more micronuclei than the negative

control, and 1% more micronuclei per nucleus than the control was considered a weak positive

result. A sample with at least 3x more micronuclei than the control, and 3% more MN per

nucleus than the negative control was considered a positive result. The highest dose saved had no

more than 55±5% cytotoxicity relative to the negative control (OECD, 2010).
                                                                            Nanosilver Toxicity 8


       The silver nanoparticles were evaluated with a long treatment time (28 hours) at

concentrations of 10–24 µg/mL in absence of S-9. The micronucleus test results are summarized

in Table 2. A preliminary experiment for dose-range finding showed similar results (data not



       Measurements of cytotoxicity used in the in vitro micronucleus assay usually combine

estimates of cell death and cytostasis. RICC, RPD and cell death have been suggested as the

most accurate cytotoxicity indicators (OECD, 2010). Graphs for these measurements are

displayed in Figures 2 and 3. Concentration-dependent decreases in cell survival were seen for

all end-points of the cytotoxicity measurements in Table 1. The dose-response for RPD and

RICC was linear (R2 = 0.985 for RPD and R2 = 0.988 for RICC) and the nanoparticles induced

significant cytotoxicity at all of the doses for both the measurements using linear trend analysis

(p < 0.001). Cell death calculated from cells with apoptosis and necrosis compared to total cells

also increased with dose, reaching to 26.4% at the highest dose while the death rates for the

negative and positive controls were 2.4% and 4.1%, respectively.


       Micronuclei were found to increase with the concentrations of silver nanoparticles

(Figure 4). In the in vitro micronucleus assay, the cytotoxicity of the top concentrations must

have no greater than 55±5% cytotoxicity for accurate micronucleus evaluation (OECD, 2010). At

a concentration of 20 µg/mL (the RPD was 59%), the fold change for the micronucleus induction

was 2.9-fold over the negative control and the net increase over the negative control was 0.69%.
                                                                             Nanosilver Toxicity 9

Therefore, the increase was not great enough to consider the nanoparticles genotoxic according

to the criteria.


        The purpose of this experiment was to discover if silver nanoparticles induce

genotoxicity and/or cytotoxicity in TK6 lymphoblast cells. A secondary purpose was to

determine whether or not current methods of assessing genotoxicity in chemicals and bulk

materials could be applied to nanoparticles. To fulfill these purposes, the in vitro micronucleus

assay was performed on silver nanoparticles (5 nm in diameter) using doses ranging from 10 - 24

µg/mL. Cytotoxicity was measured through cell counts as well as flow cytometric analysis.

        The cytotoxicity of the silver nanoparticles was shown to be highly dose-responsive

through linear trend analysis. This result is consistent with a previous study using the mouse

lymphoma assay for the same nanoparticles (Chen et al., 2010) and other studies for different

types of silver nanoparticles (Ahamed et al., 2008; Ahamed et al., 2010; AshaRani et al., 2009).

Possible reasons for high levels of cytotoxicity range from mitochondrial function being reduced,

changes in cell morphology, and oxidative stress to disruptions in the cell cycles (AshaRani, et

al., 2009).

        Several studies show that ion types and bulk types of silver are not genotoxic (Bosetti et

al., 2002; Eliopoulos and Mourelatos, 1998). However, because silver nanoparticles could be

different from other types of silver due to the effects of being nanoscale, the genotoxicity of the

nanoparticles was evaluated using the micronucleus assay. Although there was a slight linear

trend in the frequency of micronuclei in relation to dose, the test for genotoxicity in the 5 nm

silver nanoparticles was not considered positive in the TK6 cells, indicating that they did not

significantly increase chromosome damage. However, the previous experiment using the mouse
                                                                            Nanosilver Toxicity 10

lymphoma assay found that the silver nanoparticles were genotoxic and mutagenic in doses as

small as 5 µg/mL. In that study, the nanoparticle treatment for 4 hours resulted in a dose-

dependent mutation as measured using the mouse lymphoma assay. The mean mutant frequency

for treatment with 5 ng/μl nanosilver was about 7-fold higher than the untreated control. The

same treatment also produced a dose-response oxidative DNA damage in the mouse lymphoma

cells, as evaluated using oxidative Comet assay. Gene expression analysis showed that many

genes involved in production of reactive oxygen species, oxidative stress response, antioxidants

and oxygen transporters were dysregulated (Chen et al., 2010). The differences between this and

previous studies could possibly be due to the different genotoxic assays that measure different

genotoxic endpoints and/or the different cell lines - p53 proficient TK6 cells versus p53 deficient

mouse lymphoma cells. These differences could be tested in future studies.

       Other studies using different types of silver nanoparticles and different testing systems

have also resulted in a positive result for genotoxicity in silver nanoparticles. Kumari et al.

(Kumari et al., 2009) investigated genotoxicity of silver nanoparticles (< 100 nm) in Allium cepa,

a plant. They found that the nanoparticles could penetrate the plant system and impair stages of

cell division causing chromatin bridges, stickiness, disturbed metaphase, multiple chromosomal

breaks and cell disintegration. AshaRani et al. studied cytotoxicity and genotoxicity of starch-

coated silver nanoparticles using IMR-90 human lung fibroblast cells and U251 human

glioblastomas cells (AshaRani, et al., 2009). It was found that DNA damage, as measured by the

Comet assay and cytokinesis-blocked micronucleus assay, was dose dependent and more

prominent in the cancer cells. The difference between this study and other previous studies

warrant further investigation of the in vitro micronucleus assay application for assessing
                                                                            Nanosilver Toxicity 11

genotoxicity of nanomaterials. Different cell lines, such as mouse lymphoma cells could be used

as testing cells for the assays.

        In this experiment, the micronucleus test provided a non-negligible amount of

background. That is to say, the flow cytometer registered a number of unknown materials

ranging from approximately 103 to 104.25 nm in size. It is very likely that these materials were

aggregated nanosilver that, due to certain fluorescent properties, managed to register on the flow

cytometer. Although the background did not seem to affect the results, it is best to negate all

possible sources of scientific error. Therefore, future experimentation could possibly be done

using adherent cell cultures, such as the CHO cell line. Such types of cell cultures can be washed

to remove all traces of nanosilver before undergoing flow cytometry analysis. Adherent cell

cultures also have an advantage in that treatment time can be adjusted to test for negative effects

on cell cultures after exposure to nanomaterials.


        The study shows that silver nanoparticles generate a clear dose-response effect of

cytotoxicity on TK6 cells, demonstrated by multiple analyses of cytotoxicity (RPD, RICC, RCC,

etc.). However, the results from the micronuclei assay do not indicate that the nanosilver is

genotoxic in TK6 cells; they provide at best a weak positive, and more likely a negative result.

Therefore, the two-fold hypothesis is both supported and not supported.

        In the case of genotoxicity, there are various possibilities as to why the assay did not

produce positive results. As stated previously, it could be due to the cell line chosen for this

experiment. TK6 cells are known to be a relatively hardy cell line when compared to other cell

lines, such as the mouse lymphoma cell, due to various properties, such as p53 status. Another

possibility is that the nanosilver could possibly induce different types of genotoxicity, and that it
                                                                              Nanosilver Toxicity 12

is mutagenic without producing micronuclei. If it is consistently found using other assays, or cell

lines that the nanosilver is genotoxic to TK6 cells, then the micronuclei assay using TK6 cells

could possibly be not suited for testing for genotoxicity of nanoparticles.


       Firstly, I thank Dr. Robert Heflich for being my mentor, and patiently teaching me how to

properly conduct the research. Secondly, I thank Mrs. Roberta Mittelstaedt for technically

instructing me in the use of the micronucleus assay. Next, I thank NCTR for providing me with

the opportunity to conduct research at their facilities, and to use their equipment. Finally I would

like to thank my parents for their support in this project.
                                                                            Nanosilver Toxicity 13


Agarwal, A., Weis, T. L., Schurr, M. J., Faith, N. G., Czuprynski, C. J., McAnulty, J. F., Murphy,
C. J. and Abbott, N. L. (2009). Surfaces modified with nanometer-thick silver-impregnated
polymeric films that kill bacteria but support growth of mammalian cells. Biomaterials.

Ahamed, M., Karns, M., Goodson, M., Rowe, J., Hussain, S. M., Schlager, J. J. and Hong, Y.
(2008). DNA damage response to different surface chemistry of silver nanoparticles in
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Ahamed, M., Posgai, R., Gorey, T. J., Nielsen, M., Hussain, S. M. and Rowe, J. J. (2010). Silver
nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila
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AshaRani, P. V., Low Kah Mun, G., Hande, M. P. and Valiyaveettil, S. (2009). Cytotoxicity and
genotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279-90.

Bosetti, M., Masse, A., Tobin, E. and Cannas, M. (2002). Silver coated materials for external
fixation devices: in vitro biocompatibility and genotoxicity. [Abstract] Biomaterials 23, 887-92..

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toxicity. Biointerphases 2, MR17-71.

Carlson, C., Hussain, S. M., Schrand, A. M., Braydich-Stolle, L. K., Hess, K. L., Jones, R. L. and
Schlager, J. J. (2008). Unique cellular interaction of silver nanoparticles: size-dependent
generation of reactive oxygen species. J Phys Chem B 112, 13608-19.

Chen, T., Ding, W., Chen, Y., Guo, X., Zhang, Y., Biris, A., Rice, P., Ali, S., Aidoo, A., Moore,
M. and Mei, N. (2010). Genotoxicity of nanosilever in mouse lymphoma cells The Toxicologist
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Eliopoulos, P. and Mourelatos, D. (1998). Lack of genotoxicity of silver iodide in the SCE assay
in vitro, in vivo, and in the Ames/microsome test. Teratog Carcinog Mutagen 18, 303-8.

Fellows, M., Donovan M., Lorge, E., Kirkland D. (2008) Comparison of different methods for an
accurate assessment of cytotoxicity in the in vitro micronucleus test II: Practical aspects with
toxic agents. Mutation Research - Genetic Toxicology and Environmental Mutagenesis 655. 4-21

Kim, J. S., Kuk, E., Yu, K. N., Kim, J. H., Park, S. J., Lee, H. J., Kim, S. H., Park, Y. K., Park, Y.
H., Hwang, C. Y., Kim, Y. K., Lee, Y. S., Jeong, D. H. and Cho, M. H. (2007). Antimicrobial
effects of silver nanoparticles. Nanomedicine 3, 95-101.

Kim, K. J., Sung, W. S., Moon, S. K., Choi, J. S., Kim, J. G. and Lee, D. G. (2008). Antifungal
effect of silver nanoparticles on dermatophytes. J Microbiol Biotechnol 18, 1482-4.

Kim, S. W., Kim, K. S., Lamsal, K., Kim, Y. J., Kim, S. B., Jung, M., Sim, S. J., Kim, H. S.,
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Kumari, M., Mukherjee, A. and Chandrasekaran, N. (2009). Genotoxicity of silver nanoparticles
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(2008). Silver nanoparticles inhibit hepatitis B virus replication. Antivir Ther 13, 253-62.

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                                                                     Nanosilver Toxicity 15

Table 2. Silver nanoparticle agglomeration in different solutions

 Ag NPs solution               Particle size (nm)                   Particle charge (mV)

 Ag NP in Water                61.2± 1.6                            -9.37±0.54

 Ag NP in PBS                  1965.6±284.3                         ----------------

 Ag NP in Medium               1608.7±175.4                         -8.20±0.26
                                                                          Nanosilver Toxicity 16

Table 2. Data summary of the micronucleus test in TK6 cells treated with 5 nm silver nanoparticles.
                                                                             Cytotoxicity measurements
                   Total                                       Cell                                                       apoptotic
Code     Dose                  MN        P1 events   RCC                   RICC      Population    RPD    NBR    RNBR
                   Cells                                     Increase                                                     /necrotic
                     in      per 10000   per 10k
         µg/mL                                              in millions               doubling                              events
                  millions     cells      nuclei
  (-)      0        4.7          36        21        100%      3.6         100%         2.11       100%   4.77   100%       2.4%
 1-A       10       3.5          44        35        74%       2.4         67%          1.68       80%    3.22   67%        2.0%
 1-B       10       3.4          39        42        73%       2.3         65%          1.65       78%    3.29   69%        1.7%
 2-A       14       2.9          52        41        62%       1.8         51%          1.42       67%    2.30   48%        5.6%
 2-B       14       3.1          45        41        65%       2.0         55%          1.49       71%    2.54   53%        5.9%
 3-A       16       2.9          68        63        62%       1.8         51%          1.42       67%    2.35   49%        6.7%
 3-B       16       3.0          50        50        63%       1.9         52%          1.43       68%    2.06   43%        6.7%
 4-A       18       2.6          51        53        54%       1.5         41%          1.23       58%    1.41   30%        12.7%
 4-B       18       2.6          80        44        56%       1.5         43%          1.27       60%    1.67   35%        14.1%
 5-A       20       2.6          49        49        56%       1.5         42%          1.26       60%    1.94   41%        12.6%
 5-B       20       2.6         159         0        55%       1.5         41%          1.23       58%    0.91   19%        37.2%
 6-A       24       2.1          85        27        46%       1.0         29%          0.97       46%    1.36   28%        25.7%
 6-B       24       2.0         113        28        43%       0.9         26%          0.88       42%    0.97   20%        27.1%
 7-A     .75 gy     2.8         616        619       60%       1.7         48%          1.36       64%    2.13   45%        4.5%
 7-B     .75 gy     2.9         542        550       62%       1.8         51%          1.42       67%    2.92   61%        3.7%

Abbreviation: MN, micronucleus; RCC, relative cell count; RICC, relative increase in cell count; RPD, relative population doublings;
NBR, nuclei to bead ratio; RNBR, relative nuclei to bead ratio.
                                                                                      Nanosilver Toxicity 17

   Photograph provided by the Nanotechnology Center at the University of Arkansas at Little Rock (UALR)

Figure 1. Transmission electron microscopy images of 5 nm silver nanoparticles.
                                                                       Nanosilver Toxicity 18

   Relative Increase in Cell Count (RICC) and Relative Population Doubling (RPD) Linear
                                     Regression Analysis



                                                             RPD, R2=0.985

                                                             RICC, R2=0.988




                    0         5         10         15        20         25         30

                                       Dose (g/mL)

Figure 2. SigmaStat was used to make a linear regression analysis of RICC and RPD. The R2
values of both RICC and RPD were very close to 1, proving that there was a dose-response effect.
                                                                            Nanosilver Toxicity 19

                      Cell Death Induced by Varying Doses of 5 nm Silver Nanoparticles



Cell death (%)




                         0      10      14     16      18     20      24    X-ray

                                             Dose (g/mL)

Figure 3. Cell death was measured through flow cytometry, which allowed for the measuring of
the percentage of apoptosis/necrosis events compared to the whole.
                                                                           Nanosilver Toxicity 20

Micronuclei Induced by Varying Doses of 5 nm Silver Nanoparticles


Micronuclei in 10,000 cells






                                    0   10   14    16     18     20   24    X-Ray

                                                  Dose (g/mL)

Figure 4. The micronuclei assay was used to analyze genotoxicity of silver nanoparticles in TK6

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