DEVELOPMENT OF AN AUTOMATED
HIGH-THROUGHPUT SCREENING PROCEDURE
FOR NANOMATERIALS GENOTOXICITY
ASSESSMENT
August 2013
Acknowledgements
This research was undertaken at Flinders University, at the University of South Australia’s Mawson
Institute and at the Commonwealth Scientific and Industrial Research Organisation (CSIRO)
Animal, Food and Health Sciences (Adelaide), under commission from Safe Work Australia.
The personnel involved were Prof. Nico Voelcker, Dr. Michael Fenech, Dr. Giuseppe Vecchio, Dr.
Emily Anglin, Dr. Barbara Sanderson, Ms. Clare Cooksley, Mr. Martin Sweetman, Ms. Josefine
Splinter and Dr Howard Morris.
Funding for this proof-of-principle project was provided by the Department of Industry, Innovation,
Science and Research under the National Enabling Technologies Strategy.
The authors would like to thank Dr Vladimir Murashov (United States National Institute for
Occupational Safety and Health, NIOSH), Dr Miriam Baron (German Federal Institute for
Occupational Safety and Health, BAuA), Associate Professor Paul Wright (RMIT University), Mr
Stephen Thomas (Anglicare SA) and Dr Maxine McCall (CSIRO) for their review comments and
input on the report.
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2
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3
Executive Summary
In this project, an automated procedure for high-throughput screening of the genotoxicity of
synthetic nanoparticles has been developed.
Surface-engineered microarrays were used in order to provide in situ cell sorting, localisation, and
immobilisation of various subsets of human primary lymphocytes, suitable for on-chip bioassays.
The microarray platform was then integrated with the CBMN assay for analysis of the genotoxicity
of specific subsets of human peripheral lymphocytes exposed to nanoparticles. Protocols suitable
for automated scanning, imaging and analysis of microarrays for the assessment of micronuclei in
binucleated (BN) cells (to examine genotoxicity) and nuclear division index (NDI) (an indicator of
cytotoxicity) were developed.
The genotoxic effect of silver nanoparticles (AgNPs), with different size and surface coating, were
assessed. The genotoxic impact of nanoparticles on human B lymphocyte cell line was examined
successfully using the screening method. Preliminary investigations of primary human lymphocyte
genotoxicity has indicated that short exposure time and low nanoparticle concentrations need to be
used to examine genotoxicity effects using the CBMN assay.
The key features of the approach described are that it requires very small volumes of reagents,
allows sorting of lymphocyte subsets in situ, increases throughput of cell assays and is amenable
to high content microscopy analysis, which is important when considering the growing number of
different nanoparticle formulations already on the market and in development.
In summary, this proof-of-principle project has been successful in developing procedures for
lymphocyte separation, nanoparticle incubation, fluorescence staining and automated scoring of
microarray experiments to determine the extent of genotoxicity and make comparison between the
different nanoparticle concentrations, particle chemistries and particle sizes.
In ongoing work, the authors will use these procedures for the genotoxicity assessment of different
type of nanomaterials on human primary lymphocytes.
4
Contents
Glossary of terms
5
Background
6
Objectives
7
Key Results
8
PART 1 – Details of evaluation of cell sorting specificity on microarray
8
PART 2 – Examining cytotoxicity: Effect of concentration of different nanoparticles on
cell viability
11
PART 3 – CBMN assay after treatment of lymphocytes with silver nanoparticles
12
PART 4 – Implementation of an automated procedure for cell microarray imaging and
genotoxicity scoring
14
PART 5 – AgNP genotoxicity assessment using microarray-based high-throughput
screening
16
Discussion
23
Conclusion
25
Further work
25
References
26
Appendix 1 – Experimental protocols
27
5
Glossary of Terms
AgNPs - Silver Nanoparticles
BN - Binucleated Cells
CBMN - Cytokinesis-Block Micronucleus
CD - Cluster of Differentiation; Cluster Designation
CTRL - Control Sample
Cyt-B - Cytochalasin-B
DiO - Fluorescent Cell Membrane Stain (Vybrant™)
ENM - Engineered Nanomaterials
FBS - Fetal Bovine Serum
FDA - Fluorescein Diacetate
HPRT – Hypoxanthine Phosphorybosyl Transferase
MN - Micronuclei
MNed BN- Micronucleated Binucleated Cells
MonoN – Mononucleated Cells
MultiN – Multinucleated Cells
Nbud - Nuclear Buds
NDCI - Nuclear Division Cytotoxic Index
NDI - Nuclear Division Index
NPB - Nucleoplasmic Bridges
PBS - Phosphate-Buffered Saline
PI - Propidium Iodide
PHA - Phytohemagglutinin
PVP - Polyvinyl Pyrrolidone
SWNT - Single Walled Carbon Nanotubes
6
Background
Synthesis and commercialisation of engineered nanomaterials (ENMs) is continuing to grow due to
their particular physical and chemical characteristics and their potential applications in a wide
range of fields. The increased production of ENMs also involves potential risk for the environment
and human health and safety.
People may come into contact with nanomaterials in a number of ways. Nanomaterials may be
deliberately introduced into the human body, for example in the case of nanomaterials used in the
production of pharmaceutical formulations or otherwise used in the biomedical fields. The
probability that nanomaterials may enter into our body involuntarily is also increased also due to
their increased presence in our environment1-4.
The toxicological screening of engineered nanomaterials has not kept up with the growth in their
development and use. This situation represents a bottleneck between the synthesis of new
nanomaterials and toxicological characterisation before their commercialisation, and impacts on
capability of hazard classification of nanomaterials prior to use. Some nanomaterials potentially
have high hazards and recent findings demonstrate the presence of genotoxic and mutagenic
effects related to the administration of ENMs both in vivo and in vitro5, 6.
A number of issues impact on capability of accurately determining nanomaterial hazards.
Variations in nanotoxicity results have been observed that could be attributed to the vast number of
toxicity assays and biological model systems available to determine toxicity and the diverse
fabrication processes used to build nanoscale materials. Furthermore, the low throughput and the
high cost associated with many conventional toxicity assays also prevent the full potential of these
studies from being realised.
A solution to these problems is represented by the use of high-volume screening approaches for
the safety evaluation of ENMs. High-throughput screening, that makes use of automated platforms
to conduct assays and to generate data for their hazard ranking, has the advantage of reduced
reagent requirements, rapidity of analysis, reduced manpower needs, and providing a costeffective screening approach that can also be used to prioritize materials for in vivo testing 7, 8.
A novel high-throughput cell-based microarray platform has recently been developed, and then
successfully integrated with toxicological screening methods, including the cytokinesis-block
micronucleus (CBMN) assay. This surface engineered on-chip cytometer was used to determine
the extent and location of genetic damage present within primary human lymphocyte subsets9.
In this study, this microarray platform technology has been developed in order to develop a fully
automated analysis procedure for the screening of toxicity of different nanoparticles, in order to
design the best procedure for the genotoxicity assessment on human primary lymphocytes.
This project was commissioned by Safe Work Australia as part of the Nanotechnology Work Health
and Safety Program.
7
Objectives
The overall aim of this project is to develop an automated high-throughput screening procedure for
nanomaterials genotoxicity assessment that is capable of discriminating between the effects
induced by ENMs on different primary human lymphocyte subsets that are separated on a
microarray chip.
The specific objectives of the project are:
Objective 1. To evaluate the in situ cell sorting platforms (‘on-chip cytometer’) to study genetic
damage caused by exposure to ENM in human lymphocytes by automated analysis of CBMN
assay.
Objective 2. To develop an automated procedure for ‘scoring’ and quantifying biomarkers for
genetic damage (such as the appearance of micronuclei) on the microarray platform.
Objective 3. To evaluate the genotoxic effect induced by silver nanoparticles (AgNPs) with
different surface capping and different sizes.
8
Key Results
Experimental protocols are described in Appendix 1.
PART 1 – Details of evaluation of cell sorting specificity on microarray
In the classic CBMN assay protocol, cells are treated with cytochalasin-B (cyt-B) and then
harvested by cytocentrifugation using cytospin centrifuge. Here, in order to develop a new protocol
for the automated high-throughput screening of nanomaterials genotoxicity, we modified this
procedure introducing the step of cell separation and immobilisation onto antibody microarrays.
1.1 On-chip separation of human B and T lymphocytes
In order to develop an automated high-throughput screening procedure for nanomaterials
genotoxicity assessment that is capable of discriminating between the effect induced by ENMs in
different primary human lymphocyte subsets on a microarray platform, specificity in the cell sorting
on the microarray is needed. Cell sorting and separation is achieved by using different antibodies
to capture different cells.
Experimental details
- Anti-CD2, anti-CD20 and anti-CD45 microarray
- HR1K (human B lymphocyte cell line) and Jurkat (human T lymphocytes cell line)
- Cells collected in exponential growth phase, stained separately and mixed
- HR1K cells stained with Vybrant™ DiO (membrane), Jurkat cells stained with Hoechst
33342 (nuclei)
Three monoclonal antibodies that are capable of differentiating between human leukocytes, the
antigens CD2, CD20, and CD45, were printed in a 3x3 pattern onto epoxy-silane modified surface
of glass microscopy slides as shown in Figure 1 (top). As shown in Figure 1 (bottom), the three
different antibodies sort the B and T lymphocytes with specificity.
HR1K and Jurkat cell lines, cultured separately at density of 1x106 cells/mL were differently
stained. HR1K cells were labelled using Vybrant™ DiO Cell-labelling (specific for cell membrane),
while Jurkat cells nuclei were stained using Hoechst-33342. Equal portions (1 mL) of the stained
cell suspensions were combined and 1 mL of the mixed cell population was incubated on the
microarray. After incubation, the microarray was gently washed using PBS and cells were fixed
with formaldehyde solution at 3.7% v/v for 10 min. Images of cells separated on the microarray
were collected by Operetta® High Content Imaging System (Perkin Elmer Inc.).
Figure 1: Representative scheme of microarray slide consisting of three different antibody arrays printed in
3x3 patterns (top), and description of respective correspondence between cells and antigens (bottom).
9
Results
As shown in Figure 2, cells are present only in correspondence to the antibody pattern,
demonstrating the correct and high specificity of on-chip cell sorting. Thus, the results demonstrate
the high specificity in the capturing capability of the immobilised antibodies. B Lymphocytes (HR1K
cells) were recognised only in the correspondence of anti-CD20 and anti-CD45 microarray spots,
while T lymphocytes (Jurkat cells) were captured specifically by anti-CD2 and anti-CD45
antibodies.
Figure 2: Image of B and T lymphocytes captured on microarray. HR1K cell membranes were stained with
Vibrant™ DiO Cell-labelling (green); Jurkat cell nuclei were stained with Hoechst 33342 (blue).
1.2 Effect of cytochalasin-B (cyt-B) on separation of cells on-chip
The CBMN assay technique is based on the use of cyt-B in order to induce the cytokinesis block in
cells that allow the scoring of micronuclei in bi-nucleated (BN) cells. The frequency of micronuclei
(MN) in BN cells represents an index of genotoxicity. However, after the treatment with cyt-B,
mono-nucleated (MonoN) and multi-nucleated (MultiN) cells are also observed. Thus, it is
important to evaluate the effect of cyt-B treatment on the cells captured by antibody immobilised
onto the glass surface.
Experimental details
- HR1K (human B lymphocyte cell line) and Jurkat (human T lymphocytes cell line)
- Cell division blocked by addition of Cyt-B at 4.5 µg/mL
- Anti-CD2, anti-CD20 and anti-CD45 microarray
Figure 3 shows a representative image of HR1K cells captured onto microarray after the
cytokinesis block. As shown in Figure 3, the antibody microarray was able to capture MonoN, BN,
and MultiN cells permitting the scoring of micronuclei in BN cells.
10
Figure 3: Representative fluorescence image of HR1K cells immobilised on-chip after treatment with cyt-B.
The total number of BN cells represents an important factor in order to evaluate genotoxicity, as a
minimum of 500 BN cells must be scored. The number of microarray spots required to score for
MN using a sufficient number of BN cells needs to be determined.
The number of cells per microarray spot is shown in Figure 4. Comparing the number of untreated
(control) and cyt-B treated cells immobilised on the spots of antibodies, a 35% reduction was
observed in the number of cyt-B treated cells with respect of the untreated cells. Evaluating the
frequency of MonoN, BN and MultiN cells with respect of the total number of cells, there is about
30% of BN cells, 10% of MultiN cells and 60% of MonoN cells. Using this information, the number
of microarray spots required to score for MN, using a sufficient number of BN cells can be
calculated.
This reduction in number of cells captured is due to the increased cell footprint (area occupied) of
the cyt-B treated cells. The footprint of the lymphocyte cells is constituted mainly by the nuclei and
thus, subsequent to the block of cell division, the area each cell occupies on the chip is
approximately proportional to the number of nuclei.
Figure 4: Number of untreated (CTRL - control) and cyt-B treated HR1K cells immobilised on single
microarray spot. Data are expressed as the mean and standard error of measurement (SEM) (n = 3
experiments).
11
PART 2 – Examining cytotoxicity: Effect of concentration of different nanoparticles
on cell viability
Having determined that cells can be separated and captured on the microarray chip with
specificity, the next step was to examine the effect of different nanoparticles on cell viability, i.e. to
examine the cytotoxicity of the nanoparticles. This was undertaken for two reasons:
1. To choose suitable nanoparticles for subsequent genotoxicity testing, and
2. To determine suitable doses of nanoparticles to use for genotoxicity testing
Experimental details
- HRIK, human B lymphocyte cell line
- All NP tested at 100 and 150 µg/mL concentrations
- 24 h treatment
- Live/dead staining with fluorescein diacetate (FDA) and propidium iodide (PI)
Results
The effect of eight different, commonly used nanoparticles and 180 nm titanium dioxide particles
on the viability of the lymphocyte cell line HR1K was investigated. The particles used were 25 nm
and 180nm titanium dioxide, 10nm and 70 nm citrate-capped silver nanoparticles, 10 nm and 70
nm polyvinyl pyrrolidone- (PVP-) capped silver nanoparticles, P2 and P3 single-walled carbon
nanotubes (SWNT) and carbon black. Initial investigation looked at the effect of nanoparticle
concentration, over a 24 h exposure period. Figure 5 displays the cell viability results for all eight
nanoparticles, along with a negative media control and positive ethanol control. It can be seen in
most cases that as the nanoparticle concentration is increased from 100 to 150 µg/mL the cell
viability drops, which is expected as the amount of toxic material present is increased. There is
however no statistically significant difference in viability between the two nanoparticle
concentrations for most cases. For a number of materials, there was little difference in the cell
viability of the nanoparticle treated samples compared to the media control. The samples that
showed significantly lower cell viability to the control were the 70 nm citrate- and polyvinyl
pyrrolidone- (PVP-) capped silver nanoparticles at 150 µg/mL, along with the 10 nm citrate and
PVP silver nanoparticles at both concentrations. The 10 nm silver nanoparticles showed the
highest levels of toxicity of all the nanoparticles, with the citrate functionalised silver being more
toxic than the PVP functionalised particles.
In relation to the data on cytotoxicity and particle size, it is noted that there was little difference in
cell viability between 180 nm and 25 nm titanium dioxide particles. However, for silver
nanoparticles, the smaller size nanoparticles (10 nm) are more toxic.
12
Figure 5: Effect of nanoparticle type and concentration on lymphocyte cell viability for a 24 h exposure time.
Data are expressed as the mean and SEM (n = 5 experiments).
PART 3 – CBMN assay after treatment of lymphocytes with silver nanoparticles
Further information on cytotoxicity and an initial investigation of cell genotoxicity was undertaken
by using a CBMN assay after AgNP treatment of cells. This set of experiments did not use the
microarray chip. Instead, cells were spun onto slides.
Based on the cytotoxicity results, the 10 nm citrate-capped AgNPs were selected as the most
appropriate material for further experiments because of their pronounced cytotoxicity. A positive
genotoxic result was expected to be observed in primary lymphocytes treated with this material.
Exposure periods of 3 – 6 days were selected, with nanoparticle concentrations of 12.5 – 25 µg/mL
deemed most appropriate to induce a genotoxic response. The lower nanoparticle dosage was
chosen to reduce acute cytotoxicity effects and give cells time to potentially show signs of
genotoxicity.
Experimental details
- 10 nm citrate AgNPs
- 12.5 and 25 µg/mL concentration
- Primary human lymphocytes
- 3 and 6 day treatments
- Phytohemagglutinin (PHA) and nanoparticles were both added on day 1 for each
experiment
- Cytospin cells onto slides (not microarray)
- Cells stained with Diffquick brightfield stains
Cytotoxicity data
Primary lymphocyte cells treated with the silver nanoparticles were harvested and scored
according to the scoring protocol for the CBMN assay10. Figure 6 (a) and (b) displays the nuclear
13
division index (NDI) and the nuclear division cytotoxic index (NDCI) respectively, for the control
sample and the nanoparticle treated samples for the 3 and 6 day exposure periods.
The NDI provides a measure of the proliferative status (i.e. degree of cell division) of all viable cells
in a sample, while the NDCI is a measure of the proliferative status of viable and non-viable cells
(apoptotic and necrotic). The NDI and NDCI can be used to compare cytotoxicity between different
samples.
It can be observed that for the 3 day nanoparticle treated cell samples the NDI is around 1.2 – 1.3.
This is lower than the NDI for the control sample of approximately 1.5 and indicates that there was
a lower number of BN and MultiN cells in the nanoparticle treated samples.
This result is indicative of nanoparticle cytotoxicity impacting on lymphocyte proliferation.
However, it does not appear that the results are dependent on the dosage of AgNPs (12.5 vs. 25
µg/mL).
Cytotoxicity can be an issue when examining genotoxicity, i.e. when it comes to scoring the DNA
damage in these samples, as only viable BN cells can be used to score the damage. The problem
that can arise from the use of cytotoxic concentrations is that there may be insufficient BN cells to
score.
(a)
2
3 day NDI
6 Day NDI
2
3 Day NDCI
6 Day NDCI
1.5
Index
1.5
Index
(b)
1
0.5
1
0.5
0
0
Control
12.5 ug/mL
Treatment
25 ug/mL
Control
12.5 ug/mL
Treatment
25 ug/mL
Figure 6: Nuclear division index (NDI) (a) and nuclear division cytotoxic index (NDCI) (b), determined by
CBMN assay for 10 nm citrate AgNP treated primary lymphocytes. Data are expressed as the mean and
SEM (n = 3 experiments).
Genotoxicity data
DNA damage was scored for the 3 day sample only, as there were not enough BN cells in the 6
day sample to accurately score the damage. Figure 7 displays the observed frequency of the
markers that indicate DNA damage for the control and nanoparticle treated cells. These markers
are:
 BN cells with a micronucleus
 Total number of micronuclei
 BN cells with a nucleoplasmic bridge
 BN cells with a nuclear bud
The graph displays the frequency of markers per 1000 BN cells. Only for the 12.5 µg/mL sample,
an increase in micronuclei and multinucleated cells was observed. No significant differences in
other genotoxic indicators were observed between the three samples.
14
25
#MNed BNs
#MN in BN
Frequency / 1000 BN cells
20
#BNs with NPB
#BNs with Nbud
15
10
5
0
Control
12.5 ug/mL
25 ug/mL
Treatment
Figure 7: Frequency of DNA damage markers for 10 nm citrate silver nanoparticle treated primary
lymphocytes (BN = binucleated cell, MNed BN= micronucleated bi-nucleated cells, MN = micronuclei, NPB =
nucleoplasmic bridges, Nbud = nuclear buds). Data are expressed as the mean and SEM (n = 3
experiments).
PART 4 – Implementation of an automated procedure for cell microarray imaging
and genotoxicity scoring
Development of the procedure was initially undertaken with hydrogen peroxide, a known genotoxic
chemical, as a positive control. This experiment series focused on demonstrating the ability to
automatically acquire fluorescence microscopy images and perform automated image analysis
after the CBMN assay.
Experimental details
- Anti-CD20 microarray
- WIL2-NS (Human B lymphocyte cell line)
- Cells treated with different concentration of hydrogen peroxide
- Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)
Based on the results in Part 1, which showed that the microarray system permits the separation of
the different human lymphocytes, we used the Operetta® High Content Imaging System equipped
with Harmony software version 3.0 (Operetta imaging system, PerkinElmer, Hamburg, Germany)
for the collection of fluorescence images and automated image analysis. For proof-of-principle, we
used a microarray of anti-CD20 antibodies, the WIL2-NS cells line and hydrogen peroxide as a
genotoxic control. We chose this particular cell line for the implementation of automated analysis
procedure because it is a human B lymphoblastoid cell line that presents an excellent morphology
and a high nuclear division index due to the suppressed apoptotic activity, which is ideal for the
CBMN assay. WIL2-NS cells were treated for 1 h with different concentration of hydrogen peroxide
(0, 10, and 20 µM), then were washed by centrifugation and cultured in presence of cyt-B for 24h.
The images of cell microarrays were collected at magnification of 20x and 40x. The analysis of
microarrays was carried out using the implemented procedure described in Figure 8.
15
Figure 8: Step sequence implemented in Harmony software for the automatised analysis of genotoxicity.
Images were collected using the filter combination for the FITC and DAPI fluorescence, in order to
acquire the emission spectra of cell membrane (Vybrant) and nuclei (Hoechst 33342). After the
recognition of nuclei, cytoplasm and micronuclei, different building blocks were added for the
automatic recognition of MonoN, BN, MultiN and MNed BN cells. In the analysis block, we added
formulae to also calculate the Nuclear Division Index (NDI) and the frequency of BN cells carrying
MN.
As shown in Figure 9, the number of MN located in BN cells is increased in the cells treated with
hydrogen peroxide and the genotoxic effect is clearly related to the concentration of the genotoxic
agent. On the other hand, the different concentrations of hydrogen peroxide induce only slight
differences in the NDI, i.e. cytotoxicity is less significant. These results were validated using the
manual scoring procedure and were found to be comparable with results reported in the
literature11.
The genotoxic and cytotoxic data were as predicted for hydrogen peroxide, thus providing
validation of the procedure for cell microarray imaging and genotoxicity scoring.
16
Figure 9: Number of micronucleated binucleated (MNed BN) cells in 500 BN cells scored (top) and NDI
(bottom). Data obtained by automatised analysis of WIL2-NS cells untreated (control) and treated with
hydrogen peroxide at concentrations of 10 and 20 µM. Data are expressed as the mean and SEM (n = 3
experiments).
PART 5 – AgNP genotoxicity assessment using microarray-based high-throughput
screening
From Parts 1-4:
 Cells can be sorted on the microarray,
 Conditions for AgNP assessment were established,
 Microarray imaging and genotoxicity scoring was validated.
Using this preparatory work, genotoxicity of AgNPs was now examined using the microarray
technique.
5.1 Genotoxic effect induced by AgNPs in human B lymphocyte cell line
Experimental details
- Anti-CD20 microarray
- WIL2-NS (Human B lymphocyte cell line)
- Cells treated at 12.5 µg/mL concentration for 24 h with different AgNPs (10 and 70 nm,
citrate- and PVP-capped)
- Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)
17
WIL2-NS cells were treated with different AgNPs in order to investigate the importance of size and
surface coating in the genotoxicity assessment of this nanomaterial. In this experiment, WIL2-NS
cells were seeded at a density of 0.5x106 cells/mL in complete cell medium containing cyt-B at 4.5
μg/mL and 10 and 70 nm citrate- and PVP-capped AgNPs for 24 h. Hydrogen peroxide at 20 µM
concentration was used as positive control while untreated cells represented the negative control.
Cells were incubated on anti-CD20 microarrays, fixed and stained. Fluorescence images of cells
immobilised onto microarray slides were collected by the Operetta system and analyzed by
Harmony software following the automated procedure previously described. Results are reported in
Figure 10.
Results
As shown in the Figure 10, all the different AgNPs induced an increase in the number of BN cells
featuring MN, and in particular the highest number of MNed BN cells was observed in the cells
treated with 10 nm citrate-capped AgNPs demonstrating a strong genotoxic effect induced by
these nanoparticles. We observed that the genotoxicity induced by 10 nm citrate-capped AgNPs
after 24 h is higher that the genotoxicity induced by treatment for 1 h with 20 µM of hydrogen
peroxide, a well known genotoxic agent.11, 12
Size and surface coating play a key role in the modulation of genotoxicity induced by AgNPs.
Regarding size, the genotoxic effect induced by 70 nm citrate-capped AgNPs was about 50% less
compared to the 10 nm citrate-capped AgNPs. In terms of the nature of the surface coating, cells
treated with 10 nm PVP-capped AgNPs show about a 40% decreased number of MNed BN cells
compared to the cells treated with 10 nm citrate-capped AgNPs. Also PVP-capping had minimal
effect on the genotoxic response to 70 nm AgNPs.
Examining the relationship between NDI and micronuclei, we observed that a high number of MN
corresponded to a low NDI. Thus for the particles tested, highest genotoxicity is seen for particles
that also have the highest cytotoxicity. This is consistent with the cytotoxicity results shown in
Figure 5 in terms of the relative toxicity of the different AgNP species.
18
Figure 10: Number of BN cells featuring MN per 500 BN cells (top) and NDI (bottom) determined by
automatised analysis in WIL2-NS cells treated with AgNPs of different size (10 and 70 nm) and surface
coating (citrate- and PVP-capped). Data are expressed as the mean and SEM (n = 3 experiments).
19
5.2 Genotoxicity of AgNPs in primary human lymphocytes
Ultimately, the aim is to develop a screening tool for use with primary human lymphocytes.
Therefore, the next set of experiments investigates genotoxicity of AgNPs in primary human
lymphocytes captured and separated on the microarray chip. Primary lymphocytes often behave
differently to lymphocyte cell lines and differences between the two sets of experiments were
expected.
Experimental details
-
Anti-CD2, -CD4, -CD8, -CD20 and -CD45 microarray
Primary human lymphocytes
HR1K (human B lymphocyte cell line)
Cells treated at 12.5 µg/mL concentration for 72h with different AgNPs (10 and 70 nm,
citrate and PVP capped)
Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)
Separation of primary human lymphocytes on the microarray chip after CBMN assay
We tested the efficiency of our microarray platform in the recognition and separation of different
primary human lymphocytes by 5 different antibodies immobilised on the microarray slides. As
shown in Figure 11, microarray slides were prepared by printing 3 arrays with 3x3 patterns for each
different antibody in order to increase the number of scored cells. The microarray pattern was
specifically designed in order to permit the automatic acquisition of fluorescent images using the
348-well plate assay integrated on the Operetta system. This microarray pattern simplifies the
finding of the slide regions in which the different separated cells are located.
Figure 11: Microarray pattern: 5 different antibodies printed in 3 arrays of 3x3 spots each.
Primary lymphocyte cell division was activated by PHA and after 44 h cyt-B was added to the cell
cultures following the classical CBMN assay procedure10. Cells were incubated for 1 h onto
microarray slides and fluorescence images of cells immobilised onto antibody spots were acquired
using the Operetta system (Figure 12). Thus it was confirmed that the microarray platform can
recognise and separate primary human lymphocyte cells after performing a CBMN assay.
20
Figure 12: Representative fluorescence image of primary lymphocytes separated on microarray.
Examining the genotoxicity of AgNPs
Initial experiments
The above experiment was repeated, but now including the nanoparticle treatments in order to
establish the genotoxic effect induced by the differently sized and surface-coated AgNPs. All
nanoparticles were incubated at a concentration of 12.5 µg/mL with primary lymphocytes for 72 h.
At the same time, as a control, cells were stimulated with PHA followed by cyt-B treatment.
In this experiment, the analysis of cells immobilised onto microarray slides was impossible due to
the high cytotoxic effect induced by AgNPs. In particular, the number of BN cells was very low
(about 20%), the presence of nuclear debris, and cells with morphologically-deformed nuclei
caused several problems in the recognition of nuclei, micronuclei and cells immobilised onto the
microarray.
In Figure 13, representative images collected by Operetta system of anti-CD45 spots are shown.
As shown in Figure 13A, primary human lymphocytes treated with PHA and cyt-B (control) present
a good morphology of nuclei with a high presence of BN and MultiN cells. Figure 13B and Figure
13C are human primary lymphocytes treated with 12.5 µg/mL of 10 nm citrate- and PVP-capped
AgNPs respectively, and show the presence of nuclear debris and morphologically-malformed
nuclei. The same effects were observed in cells treated with 70 nm citrate- and PVP-capped
AgNPs.
This result was probably due to the previously observed cytotoxic effect induced by AgNPs.
Apoptotic and necrotic events can induce the presence of cellular and nuclear debris and it is well
known that AgNPs induce changes in nuclear morphology of cells13. These last results highlighted
the necessity to adjust the concentration and the time of AgNPs exposure in order to assess the
genotoxicity of this nanomaterial by the implemented automatised procedure.
21
Figure 13: Representative fluorescence image of primary human lymphocytes on CD45 spot. A) Control; B)
10 nm citrate-capped AgNPs; C) 10 nm PVP-capped AgNPs.
Reduction in exposure time to nanoparticles
The time that cells are exposed to nanoparticles was then reduced from 72 h to 48 h (10 nm citrate
–capped AgNPs were added to cells culture 24 h after the addition of PHA) in order to limit the
extent of cytotoxic effects that could prevent BN cell formation.
22
This change in the procedure reduced the cytotoxic effect induced by AgNPs and the immobilised
cells showed a slight improvement of their aspect. Figure 14 displays two representative
microarray spots for each nanoparticle exposure time treatment of the lymphocyte cells. For each
spot, a selection of BN cells is circled to indicate the presence and proportion over the whole spot.
When the number of BN cells is compared for each spot, between the two experiments it is clear
that the time reduction of AgNPs treatment yielded a far greater percentage of BN cells. This is an
important result in terms of scoring DNA damage in the treated cells, since only BN cells can be
scored for MN formation and since a high number of scorable cells are required for accurate
scoring. However, the automated analysis was still difficult to perform in this experiment.
Figure 14: Representative fluorescence microscopy images on CD45 microarray spots of lymphocytes
treated for 72 h (A) and 48 h (B). Cells stained with Cell Tracker Orange (cytoplasm) and Hoechst 33342
(nuclei). BN cells are circled in yellow.
Reduction in nanoparticle concentration
In subsequent experiments, we examined the use of HR1K cells with a 100-fold lower
concentration of 10 nm citrate-capped AgNPs at 48 h exposure time. HR1K is a cell line but is
more similar in behaviour to primary lymphocytes than WIL2-NS.
In this case, 10 nm citrate- and PVP-capped AgNPs at final concentration of 12.5 µg/mL (as
previously) and 125 ng/mL were used. As previously, the automated analysis of micronuclei was
difficult to perform for the cells treated with 12.5 µg/mL, due to the presence of cellular and nuclear
debris and the presence of morphologically deformed cells. However, cells treated with AgNPs
diluted by a factor of 100 to 125 ng/mL show a good morphological aspect and the presence of
nuclear and cellular debris were comparable to the control.
The automated analysis of MN of this sample revealed the persisting presence of genotoxicity
induced by the differently capped 10 nm AgNPs at a low concentration as shown in the histogram
reported in Figure 15A, with the number of MNed BN cells being higher with citrate-capped
compared with PVP-capped AgNPs. In Figure 15B, the NDI is similar for the treatment with both
differently coated 10 nm AgNPs and significantly lower than the NDI of untreated cells. Thus both
genotoxic and cytotoxic effects are shown at this lower concentration, but the cytotoxicity is not so
severe as to prevent evaluation of genotoxicity.
Thus by decreasing both exposure time and concentration, the genotoxic effects can be examined
effectively.
23
Figure 15: Number of MNed BN cells in 1000 BN cells scored (A) and NDI (B) determined by automatised
analysis in HR1K cells treated with 125 ng/mL of 10 nm citrate- and PVP-capped AgNPs. Data are
expressed as the mean and SEM (n = 3 experiments).
Discussion
The first stage of this work confirmed that specificity in cell recognition and separation can be
achieved using antibodies in the microarray chip platform9.
Next, the effect induced by treatment of cyt-B on the number of cells immobilised onto microarray
spots was determined and a reduction in the number of cells immobilised onto microarrays was
observed. Using the mean number of cells observed per spot and the frequency of BN cells
amongst these cells, microarray slides were designed with a sufficient number of antibody spots in
order to screen at least 500 BN cells for each treatment group.
The effect of nanoparticle treatments in the specificity of cell sorting on the microarray was
examined, and it was found that incubation with nanoparticles does not seem to have an effect on
the recognition of the lymphocyte types by the printed antibodies.
Following this work, an automated procedure for nanomaterial genotoxicity assessment of cells
immobilised on-chip was developed, a key step in the development of an automated highthroughput screening platform. This new procedure was validated by evaluating the genotoxicity
induced by two different concentrations of hydrogen peroxide (10 and 20 µM), obtaining results
comparable with results reported in literature11.
Using the validated automated procedure, the genotoxicity effect induced by 4 different AgNPs (10
and 70 nm, citrate- and PVP-capped) was examined. In preliminary experiments, 10 nm citrate
capped AgNPs showed the highest levels of genotoxicity inducing a greatly increased number of
MNed BN cells with the respect of negative and positive control. Results indicated that 10 nm
citrate capped AgNPs at 12.5 µg/mL are more genotoxic than hydrogen peroxide, which was used
as the positive control at 20 µM of concentration and is well known to induce oxidative stress and
DNA damage in cells11, 12. The high toxicity induced by 10 nm citrate-capped AgNPs with respect to
other sizes is probably due to differential nanoparticle uptake and bioaccumulation in cells and it is
known that smaller nanoparticles enter cells more easily than larger ones14. Surface coatings play
a key role in the stability, uptake and toxicity of AgNPs15 and differing results were obtained for
citrate-capped and PVP-capped AgNPs.
24
Finally, the genotoxic effect induced by the four differently sized and surface-coated AgNPs in
primary human lymphocytes was assessed. Experiments demonstrated the capability of our
microarray to recognise the different primary human lymphocytes subsets after the treatment with
PHA, cyt-B and AgNPs.
At long exposure times and high concentrations of AgNPs, the automated analysis of microarray
was impossible due the presence of nuclear and cellular debris and the morphological aberration
of the treated cells. In fact, as reported in recent studies, human cells treated with AgNPs show an
increased level of apoptotic and in particular necrotic events13, as well as the presence of deformed
cells and cells with damaged membranes and large vacuolation16. Furthermore, in these studies
the genotoxic effect induced by AgNPs was evaluated by manual scoring of COMET assay and
micronucleus test, demonstrating the presence of genotoxicity induced by AgNPs.
The above result with long exposure time and high concentration of AgNPs shows that to
investigate genotoxic effects of ENM, it is important to avoid acute cytotoxicity since that impedes
the study of chromosomal abnormalities induced by genotoxicity. Preliminary results using HR1K
cells treated with a significantly lower concentration, 125 ng/mL, of the two differently coated 10 nm
AgNPs demonstrated the presence of genotoxic effects induced by this nanomaterial and the
decreased cytotoxic stress induced by the low concentration permit the automatic analysis of
samples using the Operetta system. This last observation is consistent with published works where
the persistence of DNA damage induced by AgNPs at noncytotoxic doses was demonstrated17.
This in vitro study using the high throughput screening procedure, has indicated that AgNPs may
be both cytotoxic and genotoxic. Similar results have been observed by other authors when
examining the effect on cells using in vitro methods13, 16-18
Screening tests are a first step in examining whether a material is potentially toxic and are useful in
advising whether further evaluation is needed. However, test results cannot in isolation be taken as
conclusive evidence that a material is toxic. A low concordance has previously been reported
between in vitro and in vivo toxicity for nanoparticles19.
The OECD test guidelines contain a number of tests relating to the examination of genotoxicity20.
The validity of the current OECD in vitro genotoxicity test battery for nanomaterials has been
examined and in regard to the optimum strategy required for the safety assessment of
nanomaterials, it was found there are a number outstanding questions21. However in the interim,
the authors recommend that the following approach may be used as a first-stage in vitro
assessment to screen nanomaterials for further in vivo genotoxicity evaluation:




Complete description of the physico-chemical features of the material under investigation
Mammalian cell chromosome aberration test, which could be a micronucleus assay
Mammalian cell point mutation test, e.g. HPRT assay
Extended dose responses
Further work is required on the screening methods described to establish excursion criteria for
when to take further action based on the results. The criteria would define:


No significant genotoxicity - no further testing needed
Biologically significant evidence of genotoxicity - further testing needed
In relation to the data on cytotoxicity and particle size, little difference was found in the viability of
cells exposed to 180nm and 25nm titanium dioxide particles. This contrasts with in vivo data for
titanium dioxide particles, which showed the potential for formation of lung tumours in rats was
dependent on particle size and specifically on surface area22. This is the basis of the United States
25
NIOSH’s Recommended Exposure Limits (RELs) of 0.3mg/m3 for ultrafine titanium dioxide and
2.4mg/m3 for fine-sized titanium dioxide22.
Silver is known to be potentially cytotoxic if inhaled. The Australian Workplace Exposure Standard
(8-hour Time-weighted average, TWA) for silver metal is 0.1 mg/m3 and for soluble silver
compounds is 0.01 mg/m3 (measured as Ag)23. Prolonged inhalation exposure tests in rats have
indicated that lung function changes and inflammation can occur at lower concentrations of silver
nanoparticles when compared with sub-micrometer silver particles24. In this research 10 nm sized
AgNPs were found to be more toxic than 70nm AgNPs. This finding that smaller nanoparticles
show higher toxicity in in vitro tests is in agreement with other authors14, 25.
The absence of genotoxic responses to AgNPs has been reported in a number of in vivo studies.
AgNPs did not induce genetic toxicity in male and female rat bone marrow in vivo in 28 day oral
toxicity tests26 and in 90 day inhalation toxicity tests27, and were not found to induce genotoxicity in
a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was
observed28.
This work has set the stage for automated scoring of cytogenetic damage to human lymphocytes
using synthetic nanoparticles and for determining the sub-types of lymphocytes in which the
damage is most pronounced.
Conclusion
In summary, this proof-of-principle project has been successful in developing protocols and
procedure for automated high-throughput screening on-chip genotoxicity assays of nanomaterials.
Further Work
In ongoing work, the authors will use these procedures to score microarray experiments to
determine the extent of genotoxicity and make comparison between the different incubation times,
nanoparticle concentrations, particle chemistries and particle sizes on primary human lymphocytes.
The proof-of-principle data obtained during this study has led to a grant funded by the South
Australian Government (Regione Puglia-SA Awards for Research Collaboration) to investigate the
nanotoxicity of synthetic nanoparticles produced at the Italian Institute of Technology using this
microarray platform. This work will also involve validation of the microarray technology using an in
vivo genotoxicity assay in use at the Italian Institute of Technology.
26
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27
Appendix 1
Experimental Protocols
Antibody microarray slide preparation
Glass microscopy slide surfaces were cleaned and etched by immersion for 1 h in a 3:1 mixture of
concentrated sulfuric acid (H2SO4) with hydrogen peroxide (H2O2). Subsequently, the slides were
washed thoroughly with milliQ water. After drying in an oven at 60 °C for 2 h, the substrates were
immediately modified by immersing pretreated glass slides in a solution of 10% v/v (3Glycidyloxypropyl) trimethoxysilane in anhydrous toluene for 25 min. After rinsing with toluene and
acetone several times, the modified slides were dried under nitrogen gas.
Antibody solutions at 0.2 mg/mL were printed onto the silane modified surface using XactII™
Compact Microarray System equipped with 350 µM diameter Xtend™ Capillary Microarray Pins.
Printed slides were incubated at 4 °C for 24 h in a humidified chamber, then washed with PBS and
incubated at 4°C for 24 h with a 5% solution of bovine serum albumin (BSA) in PBS. Finally, they
were washed in PBS and immediately used for cell incubation.
Cell Culture
WIL2-NS, HR1K and Jurkat cells were routinely cultivated in RPMI 1640 with 50 μM glutamine,
supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were
incubated in a humidified controlled atmosphere with a 95% to 5% ratio of air/CO 2, at 37 °C. The
medium was changed every 3 days.
Fluorescein Di-Acetate/Propidium Iodide (live/dead) Staining
375 µL of cell cultures were transferred to 1.5 mL Eppendorf tubes and centrifuged 10 min at 200
g. The cell pellet was resuspended in 50 µL of staining solution (5 mL PBS containing 5 µL FDA
(1.5 mg/mL in acetone) and 4 µL PI (20 mM in DMSO)) and incubated at room temperature for 10
min. 20 µL of stained cells were added to microscopy slide, covered with a coverslip and counted
by fluorescence microscopy.
Hydrogen peroxide treatments
One day before the CBMN assay, the WIL2-NS cells were seeded at a density of 0.3×106 cells/mL.
On the assay day, cells were washed once with HBSS by centrifugation at 180 g for 5 min. For
hydrogen peroxide exposure, cells were resuspended at a density of 0.5x106 cells/mL in HBSS
with 10 and 20 µM of hydrogen peroxide and incubated for 1 h at 37 °C in a humidified atmosphere
with 5% CO2 (CO2 incubator). Cells were then washed with HBSS by centrifugation at 180 g for 5
min and then resuspended in complete RPMI medium containing 4.5 μg/mL cyt-B and incubated
for 24 h in CO2 incubator. The experimental procedure was concluded following the steps reported
below in the “Cell immobilisation, fixation, and staining on microarray slides” section.
AgNPs treatments
AgNPs aqueous solution at 1 mg/mL of concentration (BioPure) used in this work were purchased
from nanoComposix (San Diego, CA). BioPure silver nanoparticles are fabricated and processed
using the same methods as OECD silver nanoparticle standards. AgNPs were diluted to a final
concentration of 0.1 mg/mL in RPMI 1640 with 50 μM glutamine, supplemented with 10% FBS,
100 U/mL penicillin and 100 mg/mL streptomycin. Working solutions were obtained by appropriate
dilution of this solution in complete cell medium. WIL2-NS and HR1K cells were seeded at a
density of 0.3×106 cells/mL and cultured for 24 h under normal conditions, then cells were washed
once with HBSS by centrifugation at 180 g for 5 min, resuspended at a density of 0.5x106 cells/mL
in complete RPMI medium containing 4.5 μg/mL cyt-B and AgNPs at desired concentration, and
incubated for 24 h in a CO2 incubator. The experimental procedure was concluded following the
steps reported in “Cell immobilisation, fixation, and staining on microarray slides” section.
28
Primary human lymphocytes were collected following the procedure described by M. Fenech10.
Briefly, fresh blood collected by venepuncture into vacutainer blood tubes with heparin
anticoagulant was diluted 1:1 with HBSS, gently mixed and overlayed onto Ficoll-Paque using a
ratio 1:3 (Ficoll-Paque : diluted blood). After centrifugation at 400 g for 30 min at 18-20 °C, the
leucocyte layer was collected and diluted with 3x its volume of HBSS at room temperature and
centrifuged at 180 g for 10 min at 18-22 °C. The pellet was diluted with 2x the volume removed of
HBSS and centrifuged at 100 g for 10 min at 18-20 °C. The leucocyte pellet was resuspended in 1
mL culture medium at room temperature, cells were counted and seeded at density of 1x106
cells/mL in 750 µL. Mitotic division was stimulated by adding PHA at final concentration of 30
µg/mL and cells were incubated in a CO2 incubator. AgNPs solutions were added immediately after
the adding of PHA at final concentration of 12.5 µg/mL. Cell cultures were returned to the CO2
incubator and after 44 h cyt-B was added at final concentration of 4.5 µg/mL and incubated for a
further 28 h. The experimental procedure was concluded following the steps reported in “Cell
immobilisation, fixation, and staining on microarray slides” section.
Cell immobilisation, fixation, and staining on microarray slides
After incubation for 24 h with cyt-B at 4.5 μg/mL, cells were washed with HBSS by centrifugation at
180 g for 5 min, resuspended in HBSS with 5mL Vybrant® DiO cell-labeling solution (Life
Technologies) or in a solution of CellTracker Orange (Invitrogen) at final concentration of 25 µM in
a serum-free medium, and incubated at 37°C for 15 min, then centrifuged with HBSS at 180 g for 5
min, resuspended in RPMI 1640 without FBS and incubated for 1 h on microarrays in a CO2
incubator. After incubation, microarray slides were washed with PBS to eliminate cells not captured
by antibody microarray spots, immobilised cells were fixed with 3.7% v/v formaldehyde for 10 min,
permeabilised by incubation with 0.3% v/vTriton-X 100 in PBS solution for 10 min. Finally, nuclei
were stained with 0.12 µg/mL of Hoechst 33342 in PBS, washed with PBS and then microarray
slides were allowed to dry.
Fluorescence imaging acquisition
Fluorescence images of cells immobilised on microarray slides were recorded using Operetta®
High Content Imaging System equipped with Harmony software version 3.0 (Perkin Elmer Inc.).
Automatised analysis of microarray by Harmony software
Fluorescence images acquired by Operetta system were analysed by Harmony software version
3.0. The procedure of analysis consists in the automatic detection of nuclei, cytoplasm and
micronuclei. The number of cytoplasmic regions, corresponding to the number of cells, was used to
define the number on MonoN, BN and MultiN cells by the analysis of the number of detected nuclei
in each cytoplasmic region. Finally, the number of cytoplasmic regions containing 2 nuclei and
micronuclei (>0) was categorised as MNed BN cells. In the analysis block was inserted the
formulas to calculate the index of genotoxicity (number of MNed BN cells in 500 BN cells scored)
and NDI [(MonoN cells + 2*BN Cells + 3*MultiN Cells) / (total number of cells)].
Data Analysis
Results collected from 3 or 5 different experiments were used in order to calculate the mean value
and standard error of measurement (SEM).
29