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Molecular Bioeects Branch, Air Force Research Laboratory, Wright-Patterson AFB, 2729 R Street, Bldg 837, Dayton, Ohio 45433, United States, and
Department of Chemical and Materials Engineering, University of Dayton, 300 College Park, Dayton, Ohio 45469, United States
ABSTRACT In view of the vast number of new nanomaterials (NMs) that require
testing and the constraints associated with animal models, the majority of studies to
elucidate nanotoxicological eects have occurred in vitro, with limited correlation and
applicability to in vivo systems and realistic, occupational exposure scenarios. In this study,
we developed and implemented a chronic in vitro model coupled with lower, regulatory
dosages in order to provide a more realistic assessment of NM-dependent consequences
and illuminate the implications of long-term NM exposure. When keratinocytes were
exposed to 50 nm silver nanoparticles (Ag-NPs), we determined that chronically dosed cells
operated under augmented stress and modied functionality in comparison to their acute
counterparts. Specically, Ag-NP exposure through a chronic mechanism increased p38 activation, actin disorganization, heightened ki67 expression, and
extensive gene modication. Additionally, chronic Ag-NP exposure altered the way in which cells perceived and responded to epidermal growth factor
stimulation, indicating a transformation of cell functionality. Most importantly, this study demonstrated that chronic exposure in the pg/mL range to AgNPs did not induce a cytotoxic response, but instead activated sustained stress and signaling responses, suggesting that cells are able to cope with
prolonged, low levels of Ag-NP exposure. In summary, we demonstrated that through implementation of a chronic dosimetry paradigm, which more closely
resembles realistic NM exposure scenarios, it is possible to illuminate long-term cellular consequences, which greatly dier from previously obtained acute
assessments.
KEYWORDS: chronic exposure . occupational dosage . silver nanoparticle . stress response . gene regulation . risk assessment
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* Address correspondence to
saber.hussain@us.af.mil.
Received for review July 23, 2013
and accepted March 17, 2014.
Published online March 17, 2014
10.1021/nn5009116
C 2014 American Chemical Society
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Figure 1. Timeline for chronic and acute Ag-NP exposure. At the onset of experimentation, identical vials of fresh HaCaTs
were prepared and stored. Chronically exposed cells underwent NM exposure 8 h per day, 5 days a week, for 14 weeks. After
that duration, fresh cells were prepared for acute trials and all HaCaTs underwent a 24 h NM exposure to allow for direct and
simultaneous comparison of acute and chronic dosimetry conditions. Cellular end points that targeted the stress and EGF
signaling response were then evaluated.
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from
OSHA safety
chronic dosage
acute dosage
0.4 pg/mL
4 pg/mL
400 pg/mL
28 pg/mL
280 pg/mL
28 ng/mL
soluble level
insoluble level
IDLH
10 pg/mL
100 pg/mL
10 ng/mL
end of study:
lysosomal
uid
assessment
media
media
54.3 ( 5.3
101.9 ( 3.3
9.9 ( 0.4
0.32 ( 0.04
52.8 ( 4.8
54.3
52.5 ( 5.1
142.1 ( 10.8
9.2 ( 0.2
0.62 ( 0.01
59.9 ( 7.0
47.7
336.8 ( 17.5
5.9 ( 0.4
8.89 ( 0.07
ARTICLE
TABLE
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NMs display signicantly altered behavior in physiologically relevant environments,34,39 the Ag-NPs were
recharacterized in articial lysosomal uid (ALF). Following an 8 h incubation in ALF at 37 C, evaluations of
the agglomerate size, zeta potential, and ionic dissolution of the Ag-NPs were carried out with a substantial
increase identied for NP agglomeration and dissolution rate (Table 2). Due to previous reports and the low
pH of ALF,40 these results were not surprising; however,
they are signicant, as they demonstrate the potential
for increased ion production following internalization.
Another dosimetry concern is that due to the continual proliferation of cells and the fact that the rate of
NP internalization is a function of cell cycle state,41 cells
within a culture are not necessarily exposed to equal NP
quantities. However, the daily exposure incorporated into
the design of this study should minimize this variability,
as HaCaT cells were continually exposed to Ag-NPs
throughout their cell cycle progression, normalizing the
quantity each cell was exposed to over its life cycle.
In light of the importance of dosimetry and uptake,
the ability of HaCaTs to internalize Ag-NPs over an
extended time frame was assessed quantitatively
and qualitatively through ICP-MS and CytoViva imaging, respectively (Figure 2CF). We identied small
amounts of intracellular Ag-NPs from both analyses
after 14 weeks of daily exposure, further validating the
observed degree of Ag-NP deposition and slow dissolution rate at the tested low dosages.
Long-Term Ag-NP Exposure Resulted in Augmented and
Sustained Stress Responses. First, after 14 weeks' exposure,
the HaCaTs exhibited a minimal change in the number
of viable cells (Supporting Figure 1B), with a slight
diminishment in cell number (less than 10%) associated with the 400 pg/mL condition. This response is
believed to be due to a decreased cell growth rate as a
result of elevated stress levels and not necessarily the
induction of apoptosis.42 It is also imperative to highlight the fact that at levels in the pg/mL region the
HaCaTs were able to handle sustained Ag-NP exposure
without the subsequent cytotoxic response, typically
associated with nanosilver. Next, a primary set of end
points was developed around the evaluation of recognized stress markers and included target protein
activation, actin coverage, cytokine secretion, and
modifications to gene regulation. To interpret the role
of daily 50 nm Ag-NP exposure, chronically exposed
HaCaTs were directly compared to cells that underwent a 24 h, acute exposure to a dose equivalent to the
cumulative Ag-NPs encountered by the chronic cells
(Figure 1 and Table 1). Results from these analyses
demonstrated a distinct, differential response between
chronically dosed HaCaTs at the 400 pg/mL concentration and their acute counterpart (28 ng/mL), with
chronic exposure resulting in an amplified, sustained stress
reaction (Figure 3). Furthermore, the cellular responses to
Ag-NP concentration at the 0.4 and 4 pg/mL dosages
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Figure 3. Ag-NP-induced stress response is dependent on the means of exposure. Evaluation of known cellular stress
responses was carried out following introduction of 50 nm Ag-NPs through either an acute (A-400) or chronic (C-400)
exposure scenario. (A) Confocal imaging evaluation of p38 signal transduction activation was performed through detection of
the phosphorylation state (green) and normalized by total p38 (red). Included are representative confocal images of acute
and chronically dosed cells. (B) Degree of actin (red) coverage over the cellular area was determined, and representative
images were included under acute and chronic conditions for Ag-NP introduction. (C) The number of cells that were actively
expressing ki67 (pink) and its cellular localization were evaluated in acutely and chronically dosed HaCaT cells. (D) The
quantities of secreted pro-inammatory cytokines IL-6 and TNF-R were ascertained through ELISA experimentation. Recall
that equivalent acute dosage for the 400 pg/mL condition was 28 ng/mL. For all gure sections, a two-way ANOVA with
Bonferroni post-test was run with * denoting signicance from untreated and denoting signicance from the acute at the
same concentration (n = 3, p < 0.05).
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TABLE
chronic:
chronic:
gene
CCL21
function
immunoregulation and inammatory
response
CCND1 cell cycle progression
CYP1A1 metabolism and lipid synthesis
CYP7A1 metabolism and lipid synthesis
EGR1
dierentiation and mitogenesis
GDF15 dierentiation and maintenance
GSTM3 stress response
HSPA1L stress response
HSPA8 stress response
IL6
inammatory response
PTGS1
inammatory response
RAD23A DNA damage
SERPINE1 brinolysis inhibitor
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3.37
2.56
3.62
2.05
5.72
2.61
3.71
2.94
3.31
3.15
2.45
2.23
2.40
2.50
6.09
3.69
3.60
6.12
Reported genes displayed a g2.0-fold regulation over the control (acute undosed condition) and statistical signicance. Data represent three independent trials with p < 0.05.
Empty table cells indicate that no statistically signicant change was observed.
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following EGF stimulation. The classes of genes targeted by chronic exposure are primarily signaling and
matrix/migration genes. Of particular interest is the
overexpression of Akt, MAPK, PRKCA, STAT3, and
STAT5A, which are major signal transduction mediators
and are in agreement with the observed increased EGFdependent signaling. Additionally, chronically dosed
cells displayed an over 9-fold up-regulation of MMP7,
which is downstream of p38 and responsible for breaking down the extracellular matrix.51 When taking the
previously identied increase in expression and disorganization of actin laments under consideration, it
suggests that the overexpression of MMP7 could be a
cellular defense mechanism to counteract this response.
The EGF receptor contains two primary tyrosine
sites for phosphorylation (Y1068 and Y1173). The
tyrosine site that is selectively phosphorylated dictates
the mechanism through which the cells respond to
EGF. Moreover, the balance between Y1068 and Y1173
selection has been previously shown to be susceptible
to oxidative stress.52 As the HaCaT response to EGF
stimuli was seen to be signicantly dierent for acutely
versus chronically dosed cells, it logically follows that this
tyrosine selection was also modied. For chronically
dosed cells, Y1068 was preferentially phosphorylated,
while the acutely dosed cells enhanced Y1173 phosphorylation (Supporting Information Figure 4). This
transformed means of EGF receptor activation coupled
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Figure 4. Modication to EGF-dependent Akt and Erk activation. The ability of Ag-NPs to inuence the eciency of
EGF-dependent signal transduction was evaluated through
the extent of (A) Akt phosphorylation and (B) Erk phosphorylation following stimulation with 10 ng/mL EGF. Recall that
equivalent acute dosage for the 400 pg/mL condition was
28 ng/mL. A two-way ANOVA with Bonferroni post-test was
run with * denoting signicance from untreated and
denoting signicance from the acute at the same concentration (n = 3, p < 0.05).
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EGF-dependent genes
gene
function
acute: 28 ng/mL
AKT1
AKT3
BCAR1
CCND1
FN1
MAP2K7
MMP7
PRKCA
STAT3
STAT5A
2.57
chronic: 0 pg/mL
4.27
2.22
2.90
5.06
2.04
9.40
3.94
2.08
2.13
9.86
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Reported genes displayed a g2.0-fold regulation over the control (acute undosed condition) and statistical signicance. Data represent three independent trials with
p < 0.05. Empty table cells indicate that no statistically signicant change was observed.
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Figure 6. Summary of the dierential response to Ag-NPs following execution of a chronic dosimetry delivery system. HaCaTs
that underwent a one-time, 24 h exposure to Ag-NPs displayed minimal cellular alterations. On the contrary, cells that were
exposed to the same amounts of Ag-NPs, but through a chronic exposure means, demonstrated signicant cellular
consequences, including sustained stress levels, alterations to EGF response, and modied gene expression.
METHODS
Silver Nanoparticle Characterization. Citrate-stabilized Ag-NPs
with a primary particle size of 50 nm were purchased from
nanoComposix in solution form. Verification of primary size and
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50.
51.
52.
53.
54.
55.
56.
57.
58.
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49.
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