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Article
Co-exposure of Carboxyl-functionalized Single-walled Carbon Nanotubes and 17#-ethinylestradiol in Cultured Cells: Effects on Bioactivity and Cytotoxicity Maoyong Song, Fengbang Wang, Luzhe Zeng, Junfa Yin, Hailin Wang, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 12, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Environmental Science & Technology
1
Co-exposure
of
Carboxyl-functionalized
2
Carbon Nanotubes and 17α-ethinylestradiol in Cultured Cells:
3
Effects on Bioactivity and Cytotoxicity
4
Maoyong Song*, Fengbang Wang, Luzhe Zeng, Junfa Yin, Hailin Wang, Guibin Jiang
5
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for
6
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
7
*Correspondence: [email protected] (M. Song), Tel./Fax: 0086-10-62923597
8
9
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ABSTRACT: 17α-ethinylestradiol (EE2) is the representative of environmental estrogens.
14
Although EE2 can interact with some engineered nanoparticles (NPs), little is known about
15
the bioactivity of NPs-associated EE2 in organisms. In this study, we investigated the
16
combined effects of the co-exposed carboxyl-functionalized single-walled carbon nanotubes
17
(cf-SWCNTs) and EE2 in human breast adenocarcinoma cell line (MCF-7 cells), focusing on
18
the cytotoxicity and bioactivity. There were no significant differences in mitochondrial
19
activity, membrane damage, and cell apoptosis when exposed to cf-SWCNTs with and
20
without adsorbed EE2. However, the bioactivity of adsorbed EE2 on cf-SWCNTs was
21
significantly inhibited. The calculated effective concentration of EE2 in cultured cells showed
22
that less than 0.2% of the total adsorbed EE2 was released, indicating most of the EE2
23
retained on the cf-SWCNTs during cellular exposure. Furthermore, there were no obvious
24
changes in the bioactivity of adsorbed EE2 in culture medium containing 5-20% fetal bovine
25
serum (FBS) even up to 10 day’s incubation, indicating that the adsorbed EE2 on
26
cf-SWCNTs is highly stable in cell culture medium. These results mark a promising
27
possibility for EE2 to be adsorbed by cf-SWCNTs in environmentally relevant settings, and
28
thereby influenced its toxicity and biological fate. This is also tempting for future studies
29
involving risk assessment ways for association between NPs and contaminants in the
30
environment.
31
32
KEYWORDS: Single-walled Carbon Nanotubes, EE2, Bioactivity, Cytotoxicity, Effective
33
Concentration
34 3
35
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INTRODUCTION
37
Carbon nanotubes (CNTs) have attracted a great deal of research interest because of their
38
unique mechanical, electrical, optical and biomedical properties.1, 2 In light of their increasing
39
production and potential applications, CNTs will inevitably enter the environment during
40
their manufacture, transport, handling, use and disposal. Their extremely small size, unique
41
conformation, large surface area, and propensity for surface modification raise possibility that
42
CNTs could pose a hazard to humans and other living organisms.
43
In addition to the toxicity of CNTs themselves, the interactions between CNTs and other
44
chemical contaminants in the environment may influence their environmental fate,
45
bioavailability, and toxicity. Five possible interactions leading to the adsorption of organic
46
chemicals on CNTs have been proposed: these are hydrophobic, π-π stacking, hydrogen
47
bonding, electrostatic, and covalent interactions.3-6 These strong interactions suggest that
48
CNTs should be effective adsorbents for organic chemicals in solid-phase extraction and
49
water treatment.7-9 Adsorption of natural organic matter and surfactant on CNTs can enhance
50
their stability in aqueous suspension.4, 10, 11 Furthermore, adsorbed contaminants may amplify
51
the toxicity of CNTs.12-14
52
Previous studies have shown that CNTs are capable of adsorbing organic chemicals such as
53
polycyclic aromatic hydrocarbons (PAHs),14,
15
54
phenols and anilines.5, 16-18 It is now widely accepted that adsorption onto CNTs can increase
55
the persistence of organic contaminants in the environment.19 Some evidence indicates that
56
CNT-associated contaminants are more easily taken up by organisms and thus pose a greater
17α-ethinylestradiol (EE2), bisphenol A,
4
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exposure risk. 20, 21 Concentrations of hydrophobic organic compounds (HOCs) on the surface
58
of CNTs are always many orders of magnitude higher than those in the surrounding
59
environment because of the high sorption capacities of CNTs.20, 22, 23 This means that the
60
intake of a minor amount of CNTs could bring abundant CNT-associated contaminants into
61
cells or organisms.
62
Furthermore, the observation that PAHs can reversibly adsorb onto CNTs implies that
63
CNT-associated contaminants may be bioavailable.23 Wang found that biomolecules
64
enhanced the release of residual HOCs from CNTs in an in vitro gastrointestinal tract model.
65
24
66
bioavailability to organisms.22,
67
CNT-associated contaminants is how to accurately and precisely determine their dosage,
68
toxicity, and biological effects. So far, little is known about the release and bioactivity of
69
CNT-associated contaminants after cell uptake. Therefore, it is necessary to understand the
70
adsorption and bioactivity of CNT-associated contaminants in organisms.
On the other hand, strong sorption of contaminants on CNTs can reduce their 25
A profound challenge in studying the bioactivity of
71
In the present study, EE2 was selected as a target contaminant because of its high
72
estrogenic activity at sub µg/kg concentrations.26 The objective of this study was to
73
investigate the combined effects of the association between cf-SWCNTs and EE2 in MCF-7
74
cells, with a focus on the cytotoxicity of cf-SWCNTs and the bioactivity of EE2. To
75
determine the EE2 bioactivity accurately and with high sensitivity, an MCF-7 human breast
76
carcinoma cell line, stably transfected with an ER-controlled luciferase reporter gene
77
construct (called MVLN) was used.
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MATERIALS AND METHODS Characterization
79
of
cf-SWCNTs.
Carboxyl-functionalized
single-walled
carbon
80
nanotubes (cf-SWCNTs, > 90% carbon basis, 3 wt% COOH) were purchased from
81
Sigma-Aldrich (St. Louis, MO, USA). Ten milligrams of cf-SWCNTs were suspended in 100
82
mL deionized water in a 200-mL bottle and sonicated in a water bath (KQ-250DB, 40 kHz)
83
for 1 h. The suspension was shaken on a shaker at 100 rpm for 24 h at room temperature and
84
then filtered through a membrane (pore size = 0.22 µm), followed by washing with 50 mL
85
deionized water while on the membrane. Using ultrasonication, the cf-SWCNTs were then
86
re-suspended in deionized water at a concentration of 0.5 mg/mL. The suspension of
87
cf-SWCNTs was diluted to 0.01 mg/mL, following which 5 µL of the diluent was deposited
88
on an ultrafine carbon support film on a copper grid, and dried overnight in a dust-free box.
89
The morphology and structure of cf-SWCNTs were imaged with a Hitachi H-7500
90
transmission electron microscope (TEM, Tokyo, Japan). An Agilent 7500 inductively
91
coupled plasma mass spectrometer (ICP-MS) (Santa Clara, CA, USA) was used to analyze
92
the metal impurities in cf-SWCNTs. A Zetasizer Nano (Malvern Instruments, Malvern, UK)
93
was used to measure the zeta potential (ZP) of the cf-SWCNTs in aqueous suspension. In
94
addition, Raman characterization of cf-SWCNTs was performed on an inVia Raman
95
spectroscope (Renishaw plc, Wotton-under-Edge, UK) with a 532-nm laser source.
96
Adsorption Experiment. A stock solution of 0.337 mg/L EE2 in dimethyl sulfoxide
97
(DMSO) was prepared. The adsorption experiment was carried out as described previously.5,
98
14
99
solid/solid (w/w) rations were 1:20000-1:50 for EE2/cf-SWCNT. The vials were kept in the
The initial concentrations for adsorption experiments were 6.74-3370 ng/L for EE2, and the
6
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dark and rotated vertically on a shaker at 100 rpm for 10 days to reach adsorption and
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desorption equilibrium. All experiments, including the blanks, were conducted in triplicate.
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All samples were centrifuged at 20,000 × g for 90 min, and the precipitates were collected as
103
cf-SWCNTs-EE2 composites (CSE). The amount of adsorbed EE2 in CSE was calculated
104
from the mass difference between the original and equilibrated solutions using HPLC with a
105
fluorescence detector at 250 nm (excitation wavelength) and 310 nm (emission wavelength).
106
The mobile phase was 60:40 (v:v) of acetonitrile and deionized water with 1% acetic acid,
107
and the detection limit was 6.2 ng/L. In the range of EE2 concentration from 29.6 to 14.8×103
108
ng/L, a linear correlation (r2 = 0.999) was obtained. A series of CSE (with adsorbed EE2 from
109
0.05 to 15 mg/g) were prepared in this study.
110
A solution containing 296 ng/L EE2 was subjected to identical conditions as a reference.
111
And then the stability of EE2 over 10 days was analyzed by HPLC-MS/MS. An Agilent
112
6410B Triple Quadruple mass spectrometer (Santa Clara, CA, USA) with an electrospray
113
ionization source was applied for mass spectrometer detection. Mass spectrometer conditions
114
were optimized as follows: ionization mode, ESI-negative; capillary voltage, 4000 V;
115
nitrogen drying gas temperature, 300 °C; drying gas flow, 9 L/min; nebulizer, 40 psi. For
116
MS/MS analysis of EE2, the fragmentor voltage was 150 V, collision energy was performed
117
at 40 eV, and scan time was 100 ms.
118
Cytotoxicity Assays. MCF-7 cells were used for all cytotoxicity assays. Cell viability was
119
assessed by an aqueous soluble tetrazolium/formazan assay (MTS assay) based on the
120
conversion of a tetrazolium salt into a colored, water-soluble formazan product by
121
mitochondrial activity.27 After 24 h of cell attachment, cells were exposed to the tested 7
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samples at series concentrations. DMSO was added to three wells as a negative control (NC).
123
Twenty-four hours later, the cells were incubated with the MTS assay reagents for 4 h, and
124
absorbance was measured at 490 nm by a microplate reader (Varioskan Flash, Thermo, USA).
125
Cell viability was expressed as a percent of NC value.
126
Damage to the plasma membrane was evaluated by measuring lactate dehydrogenase
127
(LDH) leakage from the cytosolic phase to the medium.28 In brief, after 24 h exposure to
128
samples, the medium was removed from the culture cell, then incubated with the test reagent
129
resazurin (CytoTox-ONETM Homogeneous Membrane Integrity Assay, Promega, Madison,
130
WI, USA) at 22 °C for 10 min, following which the fluorescence of the medium was
131
determined by a microplate reader (Varioskan Flash, Thermo). Upon its release into the
132
solution, the LDH converted resazurin into resorufin, whose emission at 590 nm can be
133
detected upon 560 nm excitation. Positive control did not receive any treatment (PC). LDH
134
leakage was calculated as the percentage of LDH in the medium versus total LDH activity of
135
PC.
136
Apoptotic and necrotic cells were analyzed by double staining with annexin V-FITC
137
(annexin V coupled to fluorein isothiocyanate) and PI (propidium iodide), which were both
138
obtained from Molecular Probes, USA. The stained cells were then counted by flow
139
cytometry. In brief, after 24 h exposure to test samples, cells were harvested by trypsinization
140
and pipetting up and down numerous times, washed once with cold PBS, and pelleted via
141
centrifugation at 200 × g for 10 min. For each sample, 5 mL of annexin V-FITC was added to
142
400 mL of cells suspended in binding buffer, which were then incubated for 15 min at room
143
temperature in the dark. Immediately afterwards, the cells were stained with 10 mL PI and 8
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analyzed by a flow cytometer (LSRII, BD Biosciences, USA). Cells treated with DMSO was
145
analyzed as a negative control (NC).
146
MVLN Assay. The relative estrogenic activity was determined by the MVLN assay. The
147
MVLN cell line was kindly provided by J. P. Giesy (Michigan State University, East Lansing,
148
MI, USA). This cell line was stably transfected with the luciferase reporter-gene and
149
estrogen-responsive element, so that estrogen receptor (ER) agonists would induce the cells
150
to produce luciferase. Estrogenic activity of each sample was determined as previously
151
described.29 In brief, 60 interior wells of a 96-well culture plate were each seeded with 250
152
µL cell suspension at a density of about 50,000 cells per well. The cells were starved in
153
steroid-free medium for 48 h under aseptic conditions in a humidified CO2 incubator at 37 °C
154
and 5% CO2 before exposure. 17β-estradiol (E2, 98%) (Sigma) was used as a positive control,
155
and six dilutions of E2 were prepared. The dilutions were then added to three wells per
156
dilution leading to the final E2 concentration of 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 nM.
157
Sample solutions were diluted to the same concentrations and added to the sample wells in
158
the same way. As a negative control, solvent was added to three wells. The cells were then
159
incubated for 48 h in a humidified CO2 incubator at 37 °C and 5% CO2 until the luciferase
160
assay was carried out. After removal of the culture medium, cells were washed three times
161
with phosphate-buffered saline (PBS) buffer, and then lysed with 75 µL PBS buffer
162
containing Ca2+ and Mg2+, following which 75 µL luciferase assay reagent was added to each
163
well, and the plates were incubated for 20 min at room temperature in the dark. The
164
luminence was detected by a microplate reader (Varioskan Flash, Thermo) and normalized by
165
the results of a Bradford assay, which was simultaneously carried out to determine total 9
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protein content. The maximal luciferase activity induction of E2 was set as 100%, and the
167
responses of other samples were converted to a percentage of the maximum level.
168
Statistical analysis. SPSS statistical software (Version 13.0) and Sigma Plot 10.0 were
169
used for statistical analysis. The significant differences between control and treated groups
170
were determined using a one-way analysis of variance and Tukey’s multiple range test.
171
Differences were statistically significant if p < 0.05.
172
RESULTS AND DISCUSSION
173
Characterization of cf-SWCNTs and CSE. In the Raman spectrum of cf-SWCNTs, two
174
peaks are visible that correspond to the G-band at 1593 cm-1, derived from the graphite
175
structure, and the D-band at 1346 cm-1 derived from defects (Figure 1A). The levels of metal
176
impurities in cf-SWCNTs were found to be 0.07 wt% iron and 0.16 wt% nickel. Cobalt and
177
manganese were not detected ( 0.05). The
207
cytotoxicity of CSE with higher concentrations of adsorbed EE2 was not further investigated
208
because higher concentrations would not have been environmentally relevant. 11
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Estrogenic Activity of Adsorbed EE2. Cf-SWCNTs only displayed no estrogenic activity
210
at concentrations from 5 to 40 µg/mL (data not shown). As shown in Figure 3A, the relative
211
estrogenic activity of EE2 control (final exposure concentration was 29.6 ng/L) was 91%.
212
However, the relative estrogenic activity of CSE-1 (with 0.11 mg/g adsorbed EE2) was from
213
22.1 to 55.1% when the final exposure concentration of adsorbed EE2 was from 29.6 to 592
214
ng/L. The relative estrogenic activities of CSE-2 (with 11 mg/g adsorbed EE2) were 75.5%
215
and 99.1% when the final exposure concentration of adsorbed EE2 were 2.96×103 and
216
1.48×104 ng/L, respectively. We investigated the stability of EE2 over 10-day incubation
217
using HPLC-MS and MVLN assays. There were no significant changes in EE2 quality and
218
estrogenic activity after 10-day incubation followed by centrifugation (20,000 × g) comparing
219
with fresh EE2 (Figure S1), indicating that there was no significant mass loss or degradation
220
during exposure. These results indicated that the bioactivity of EE2 was significantly reduced
221
by the adsorption of cf-SWCNTs.
222
While it is known that the free EE2 are capable of inducing expression of luciferase in the
223
MVLN assay, no previous effort has been made to clarify the luciferase inductivity of
224
adsorbed EE2 on NPs. We supposed the luciferase induction is due to the EE2 desorbed from
225
cf-SWCNTs. However, analytical determination of free EE2 in cells would be extremely
226
difficult because of the low concentrations involved, which would require chemical
227
extraction from large quantities of cultured cells. Moreover, methods such as solvent
228
extraction would destroy the association between EE2 and cf-SWCNTs during extraction.
229
The response of MVLN cells is highly sensitive and can indicate the estrogenic activity of
230
EE2 at pg/L levels. In order to measure the EE2 desorption during exposure, a linear 12
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correlation (r2 = 0.997) in the range of EE2 concentration from 2.96 × 10-2 to 2.96 ng/L was
232
obtained (Figure 3B). Therefore, within this range, the EE2 concentration that induced
233
expression of luciferase, named effective concentration, can be calculated. So we calculated
234
the effective concentrations of adsorbed EE2 during exposure. For example, the final
235
exposure concentration of EE2 for CSE-1 was 592 ng/L, but its effective concentration of
236
EE2 was calculated as 0.89 ng/L, indicating that only 0.15% of the adsorbed EE2 was
237
desorbed from cf-SWCNTs. As shown in Figure 3C, the desorption rates of all test groups
238
(estimated from the effective concentration and adsorption concentration) were less than
239
0.2%, indicating most EE2 remained on cf-SWCNTs during exposure.
240
It has been reported that fullerene (C60) co-exposed with benzo[α]pyrene (Bap) increases
241
Bap accumulation in cells.30 The uptake of cf-SWCNTs during phagocytosis, diffusion and
242
endocytosis31 may facilitate the transport of EE2 through cell membranes, then influenced the
243
bioactivity of EE2. However, little is known whether EE2 could be adsorbed onto
244
cf-SWCNTs in cell culture medium during co-exposure, and then influenced its bioactivity.
245
Here cf-SWCNTs were added to the cell culture medium containing EE2, then the estrogenic
246
activity of EE2 was measured after 48 h exposure. There were no significant changes in the
247
relative estrogenic activity between all cf-SWCNTs+EE2 groups and EE2 control (29.6 ng/L)
248
(p > 0.05) (Figure S2A), indicating the bioactivity of EE2 was not inhibited by additional
249
cf-SWCNTs. We also measured the estrogenic activities of cf-SWCNTs+EE2 groups and
250
EE2 control every 12 h during exposure, and showed a similar tendency towards variation in
251
estrogenic activity (Figure S2B), indicating that the response speed of EE2 were not
252
influenced by additional cf-SWCNTs during co-exposure. Because it is known that a number 13
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of proteins (e.g., serum albumin, immunoglobulin, streptavidin) are capable of nonspecific
254
binding to carbon nanotube surface,32 that EE2 could not adsorbed onto cf-SWCNTs during
255
co-exposure may be due to the competitive adsorption of proteins in cell culture medium.
256
Competitive adsorption of PAHs with other organic chemicals such as dissolved organic
257
matter and surfactants has previously been studied.33, 34 Xing et al. reported that the release of
258
residual HOC from CNTs could be enhanced by biomolecules such as pepsin and bile salts in
259
the digestive tract, thus increasing the bioaccessibility of adsorbed phenanthrene and possibly
260
the overall toxicity of phenanthrene associated with CNTs.24 It is unknown whether the
261
biomolecules in cell culture or cytoplasm could lead to the desorption of EE2 from
262
cf-SWCNTs. Here we investigated the bioactivity change of adsorbed EE2 in culture medium
263
containing different concentrations of FBS under extended incubation time. As shown in
264
Figure 4, there were no obvious changes in the estrogenic activity of adsorbed EE2 in culture
265
medium containing 5-20% FBS over 10 day’s incubation. It suggests that the adsorbed EE2
266
of cf-SWCNTs is highly stable in cell culture medium. However, the bioactivity of adsorbed
267
EE2 on cf-SWCNTs may be prolonged and cause chronic situation in biological or
268
environmental relevant setting because of its high stability and possible desorption.
269
Desorption of toxic chemicals from CNTs is very important for evaluating the
270
environmental and health risk of chemicals, because desorption renders these chemicals
271
mobile and bioavailable in the environment or in organisms. Previous study has shown
272
reversible adsorption of PAHs on CNTs.23 However, irreversible hysteresis has been
273
observed for some organic chemicals such as bisphenol A and EE2 upon their desorption
274
from CNTs into water.5 It has been proposed that the irreversible hysteresis of EE2 from 14
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CNTs was attributed to the rearrangement of bundles or aggregates of CNTs.5, 35 However, a
276
few adsorbed EE2 (
Article
Co-exposure of Carboxyl-functionalized Single-walled Carbon Nanotubes and 17#-ethinylestradiol in Cultured Cells: Effects on Bioactivity and Cytotoxicity Maoyong Song, Fengbang Wang, Luzhe Zeng, Junfa Yin, Hailin Wang, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 12, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
Environmental Science & Technology
1
Co-exposure
of
Carboxyl-functionalized
2
Carbon Nanotubes and 17α-ethinylestradiol in Cultured Cells:
3
Effects on Bioactivity and Cytotoxicity
4
Maoyong Song*, Fengbang Wang, Luzhe Zeng, Junfa Yin, Hailin Wang, Guibin Jiang
5
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for
6
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
7
*Correspondence: [email protected] (M. Song), Tel./Fax: 0086-10-62923597
8
9
1
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TOC Figure
11 12
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ABSTRACT: 17α-ethinylestradiol (EE2) is the representative of environmental estrogens.
14
Although EE2 can interact with some engineered nanoparticles (NPs), little is known about
15
the bioactivity of NPs-associated EE2 in organisms. In this study, we investigated the
16
combined effects of the co-exposed carboxyl-functionalized single-walled carbon nanotubes
17
(cf-SWCNTs) and EE2 in human breast adenocarcinoma cell line (MCF-7 cells), focusing on
18
the cytotoxicity and bioactivity. There were no significant differences in mitochondrial
19
activity, membrane damage, and cell apoptosis when exposed to cf-SWCNTs with and
20
without adsorbed EE2. However, the bioactivity of adsorbed EE2 on cf-SWCNTs was
21
significantly inhibited. The calculated effective concentration of EE2 in cultured cells showed
22
that less than 0.2% of the total adsorbed EE2 was released, indicating most of the EE2
23
retained on the cf-SWCNTs during cellular exposure. Furthermore, there were no obvious
24
changes in the bioactivity of adsorbed EE2 in culture medium containing 5-20% fetal bovine
25
serum (FBS) even up to 10 day’s incubation, indicating that the adsorbed EE2 on
26
cf-SWCNTs is highly stable in cell culture medium. These results mark a promising
27
possibility for EE2 to be adsorbed by cf-SWCNTs in environmentally relevant settings, and
28
thereby influenced its toxicity and biological fate. This is also tempting for future studies
29
involving risk assessment ways for association between NPs and contaminants in the
30
environment.
31
32
KEYWORDS: Single-walled Carbon Nanotubes, EE2, Bioactivity, Cytotoxicity, Effective
33
Concentration
34 3
35
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INTRODUCTION
37
Carbon nanotubes (CNTs) have attracted a great deal of research interest because of their
38
unique mechanical, electrical, optical and biomedical properties.1, 2 In light of their increasing
39
production and potential applications, CNTs will inevitably enter the environment during
40
their manufacture, transport, handling, use and disposal. Their extremely small size, unique
41
conformation, large surface area, and propensity for surface modification raise possibility that
42
CNTs could pose a hazard to humans and other living organisms.
43
In addition to the toxicity of CNTs themselves, the interactions between CNTs and other
44
chemical contaminants in the environment may influence their environmental fate,
45
bioavailability, and toxicity. Five possible interactions leading to the adsorption of organic
46
chemicals on CNTs have been proposed: these are hydrophobic, π-π stacking, hydrogen
47
bonding, electrostatic, and covalent interactions.3-6 These strong interactions suggest that
48
CNTs should be effective adsorbents for organic chemicals in solid-phase extraction and
49
water treatment.7-9 Adsorption of natural organic matter and surfactant on CNTs can enhance
50
their stability in aqueous suspension.4, 10, 11 Furthermore, adsorbed contaminants may amplify
51
the toxicity of CNTs.12-14
52
Previous studies have shown that CNTs are capable of adsorbing organic chemicals such as
53
polycyclic aromatic hydrocarbons (PAHs),14,
15
54
phenols and anilines.5, 16-18 It is now widely accepted that adsorption onto CNTs can increase
55
the persistence of organic contaminants in the environment.19 Some evidence indicates that
56
CNT-associated contaminants are more easily taken up by organisms and thus pose a greater
17α-ethinylestradiol (EE2), bisphenol A,
4
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exposure risk. 20, 21 Concentrations of hydrophobic organic compounds (HOCs) on the surface
58
of CNTs are always many orders of magnitude higher than those in the surrounding
59
environment because of the high sorption capacities of CNTs.20, 22, 23 This means that the
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intake of a minor amount of CNTs could bring abundant CNT-associated contaminants into
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cells or organisms.
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Furthermore, the observation that PAHs can reversibly adsorb onto CNTs implies that
63
CNT-associated contaminants may be bioavailable.23 Wang found that biomolecules
64
enhanced the release of residual HOCs from CNTs in an in vitro gastrointestinal tract model.
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24
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bioavailability to organisms.22,
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CNT-associated contaminants is how to accurately and precisely determine their dosage,
68
toxicity, and biological effects. So far, little is known about the release and bioactivity of
69
CNT-associated contaminants after cell uptake. Therefore, it is necessary to understand the
70
adsorption and bioactivity of CNT-associated contaminants in organisms.
On the other hand, strong sorption of contaminants on CNTs can reduce their 25
A profound challenge in studying the bioactivity of
71
In the present study, EE2 was selected as a target contaminant because of its high
72
estrogenic activity at sub µg/kg concentrations.26 The objective of this study was to
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investigate the combined effects of the association between cf-SWCNTs and EE2 in MCF-7
74
cells, with a focus on the cytotoxicity of cf-SWCNTs and the bioactivity of EE2. To
75
determine the EE2 bioactivity accurately and with high sensitivity, an MCF-7 human breast
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carcinoma cell line, stably transfected with an ER-controlled luciferase reporter gene
77
construct (called MVLN) was used.
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MATERIALS AND METHODS Characterization
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of
cf-SWCNTs.
Carboxyl-functionalized
single-walled
carbon
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nanotubes (cf-SWCNTs, > 90% carbon basis, 3 wt% COOH) were purchased from
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Sigma-Aldrich (St. Louis, MO, USA). Ten milligrams of cf-SWCNTs were suspended in 100
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mL deionized water in a 200-mL bottle and sonicated in a water bath (KQ-250DB, 40 kHz)
83
for 1 h. The suspension was shaken on a shaker at 100 rpm for 24 h at room temperature and
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then filtered through a membrane (pore size = 0.22 µm), followed by washing with 50 mL
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deionized water while on the membrane. Using ultrasonication, the cf-SWCNTs were then
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re-suspended in deionized water at a concentration of 0.5 mg/mL. The suspension of
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cf-SWCNTs was diluted to 0.01 mg/mL, following which 5 µL of the diluent was deposited
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on an ultrafine carbon support film on a copper grid, and dried overnight in a dust-free box.
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The morphology and structure of cf-SWCNTs were imaged with a Hitachi H-7500
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transmission electron microscope (TEM, Tokyo, Japan). An Agilent 7500 inductively
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coupled plasma mass spectrometer (ICP-MS) (Santa Clara, CA, USA) was used to analyze
92
the metal impurities in cf-SWCNTs. A Zetasizer Nano (Malvern Instruments, Malvern, UK)
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was used to measure the zeta potential (ZP) of the cf-SWCNTs in aqueous suspension. In
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addition, Raman characterization of cf-SWCNTs was performed on an inVia Raman
95
spectroscope (Renishaw plc, Wotton-under-Edge, UK) with a 532-nm laser source.
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Adsorption Experiment. A stock solution of 0.337 mg/L EE2 in dimethyl sulfoxide
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(DMSO) was prepared. The adsorption experiment was carried out as described previously.5,
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14
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solid/solid (w/w) rations were 1:20000-1:50 for EE2/cf-SWCNT. The vials were kept in the
The initial concentrations for adsorption experiments were 6.74-3370 ng/L for EE2, and the
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dark and rotated vertically on a shaker at 100 rpm for 10 days to reach adsorption and
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desorption equilibrium. All experiments, including the blanks, were conducted in triplicate.
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All samples were centrifuged at 20,000 × g for 90 min, and the precipitates were collected as
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cf-SWCNTs-EE2 composites (CSE). The amount of adsorbed EE2 in CSE was calculated
104
from the mass difference between the original and equilibrated solutions using HPLC with a
105
fluorescence detector at 250 nm (excitation wavelength) and 310 nm (emission wavelength).
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The mobile phase was 60:40 (v:v) of acetonitrile and deionized water with 1% acetic acid,
107
and the detection limit was 6.2 ng/L. In the range of EE2 concentration from 29.6 to 14.8×103
108
ng/L, a linear correlation (r2 = 0.999) was obtained. A series of CSE (with adsorbed EE2 from
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0.05 to 15 mg/g) were prepared in this study.
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A solution containing 296 ng/L EE2 was subjected to identical conditions as a reference.
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And then the stability of EE2 over 10 days was analyzed by HPLC-MS/MS. An Agilent
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6410B Triple Quadruple mass spectrometer (Santa Clara, CA, USA) with an electrospray
113
ionization source was applied for mass spectrometer detection. Mass spectrometer conditions
114
were optimized as follows: ionization mode, ESI-negative; capillary voltage, 4000 V;
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nitrogen drying gas temperature, 300 °C; drying gas flow, 9 L/min; nebulizer, 40 psi. For
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MS/MS analysis of EE2, the fragmentor voltage was 150 V, collision energy was performed
117
at 40 eV, and scan time was 100 ms.
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Cytotoxicity Assays. MCF-7 cells were used for all cytotoxicity assays. Cell viability was
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assessed by an aqueous soluble tetrazolium/formazan assay (MTS assay) based on the
120
conversion of a tetrazolium salt into a colored, water-soluble formazan product by
121
mitochondrial activity.27 After 24 h of cell attachment, cells were exposed to the tested 7
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samples at series concentrations. DMSO was added to three wells as a negative control (NC).
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Twenty-four hours later, the cells were incubated with the MTS assay reagents for 4 h, and
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absorbance was measured at 490 nm by a microplate reader (Varioskan Flash, Thermo, USA).
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Cell viability was expressed as a percent of NC value.
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Damage to the plasma membrane was evaluated by measuring lactate dehydrogenase
127
(LDH) leakage from the cytosolic phase to the medium.28 In brief, after 24 h exposure to
128
samples, the medium was removed from the culture cell, then incubated with the test reagent
129
resazurin (CytoTox-ONETM Homogeneous Membrane Integrity Assay, Promega, Madison,
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WI, USA) at 22 °C for 10 min, following which the fluorescence of the medium was
131
determined by a microplate reader (Varioskan Flash, Thermo). Upon its release into the
132
solution, the LDH converted resazurin into resorufin, whose emission at 590 nm can be
133
detected upon 560 nm excitation. Positive control did not receive any treatment (PC). LDH
134
leakage was calculated as the percentage of LDH in the medium versus total LDH activity of
135
PC.
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Apoptotic and necrotic cells were analyzed by double staining with annexin V-FITC
137
(annexin V coupled to fluorein isothiocyanate) and PI (propidium iodide), which were both
138
obtained from Molecular Probes, USA. The stained cells were then counted by flow
139
cytometry. In brief, after 24 h exposure to test samples, cells were harvested by trypsinization
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and pipetting up and down numerous times, washed once with cold PBS, and pelleted via
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centrifugation at 200 × g for 10 min. For each sample, 5 mL of annexin V-FITC was added to
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400 mL of cells suspended in binding buffer, which were then incubated for 15 min at room
143
temperature in the dark. Immediately afterwards, the cells were stained with 10 mL PI and 8
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analyzed by a flow cytometer (LSRII, BD Biosciences, USA). Cells treated with DMSO was
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analyzed as a negative control (NC).
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MVLN Assay. The relative estrogenic activity was determined by the MVLN assay. The
147
MVLN cell line was kindly provided by J. P. Giesy (Michigan State University, East Lansing,
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MI, USA). This cell line was stably transfected with the luciferase reporter-gene and
149
estrogen-responsive element, so that estrogen receptor (ER) agonists would induce the cells
150
to produce luciferase. Estrogenic activity of each sample was determined as previously
151
described.29 In brief, 60 interior wells of a 96-well culture plate were each seeded with 250
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µL cell suspension at a density of about 50,000 cells per well. The cells were starved in
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steroid-free medium for 48 h under aseptic conditions in a humidified CO2 incubator at 37 °C
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and 5% CO2 before exposure. 17β-estradiol (E2, 98%) (Sigma) was used as a positive control,
155
and six dilutions of E2 were prepared. The dilutions were then added to three wells per
156
dilution leading to the final E2 concentration of 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 nM.
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Sample solutions were diluted to the same concentrations and added to the sample wells in
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the same way. As a negative control, solvent was added to three wells. The cells were then
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incubated for 48 h in a humidified CO2 incubator at 37 °C and 5% CO2 until the luciferase
160
assay was carried out. After removal of the culture medium, cells were washed three times
161
with phosphate-buffered saline (PBS) buffer, and then lysed with 75 µL PBS buffer
162
containing Ca2+ and Mg2+, following which 75 µL luciferase assay reagent was added to each
163
well, and the plates were incubated for 20 min at room temperature in the dark. The
164
luminence was detected by a microplate reader (Varioskan Flash, Thermo) and normalized by
165
the results of a Bradford assay, which was simultaneously carried out to determine total 9
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protein content. The maximal luciferase activity induction of E2 was set as 100%, and the
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responses of other samples were converted to a percentage of the maximum level.
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Statistical analysis. SPSS statistical software (Version 13.0) and Sigma Plot 10.0 were
169
used for statistical analysis. The significant differences between control and treated groups
170
were determined using a one-way analysis of variance and Tukey’s multiple range test.
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Differences were statistically significant if p < 0.05.
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RESULTS AND DISCUSSION
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Characterization of cf-SWCNTs and CSE. In the Raman spectrum of cf-SWCNTs, two
174
peaks are visible that correspond to the G-band at 1593 cm-1, derived from the graphite
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structure, and the D-band at 1346 cm-1 derived from defects (Figure 1A). The levels of metal
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impurities in cf-SWCNTs were found to be 0.07 wt% iron and 0.16 wt% nickel. Cobalt and
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manganese were not detected ( 0.05). The
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cytotoxicity of CSE with higher concentrations of adsorbed EE2 was not further investigated
208
because higher concentrations would not have been environmentally relevant. 11
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Estrogenic Activity of Adsorbed EE2. Cf-SWCNTs only displayed no estrogenic activity
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at concentrations from 5 to 40 µg/mL (data not shown). As shown in Figure 3A, the relative
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estrogenic activity of EE2 control (final exposure concentration was 29.6 ng/L) was 91%.
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However, the relative estrogenic activity of CSE-1 (with 0.11 mg/g adsorbed EE2) was from
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22.1 to 55.1% when the final exposure concentration of adsorbed EE2 was from 29.6 to 592
214
ng/L. The relative estrogenic activities of CSE-2 (with 11 mg/g adsorbed EE2) were 75.5%
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and 99.1% when the final exposure concentration of adsorbed EE2 were 2.96×103 and
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1.48×104 ng/L, respectively. We investigated the stability of EE2 over 10-day incubation
217
using HPLC-MS and MVLN assays. There were no significant changes in EE2 quality and
218
estrogenic activity after 10-day incubation followed by centrifugation (20,000 × g) comparing
219
with fresh EE2 (Figure S1), indicating that there was no significant mass loss or degradation
220
during exposure. These results indicated that the bioactivity of EE2 was significantly reduced
221
by the adsorption of cf-SWCNTs.
222
While it is known that the free EE2 are capable of inducing expression of luciferase in the
223
MVLN assay, no previous effort has been made to clarify the luciferase inductivity of
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adsorbed EE2 on NPs. We supposed the luciferase induction is due to the EE2 desorbed from
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cf-SWCNTs. However, analytical determination of free EE2 in cells would be extremely
226
difficult because of the low concentrations involved, which would require chemical
227
extraction from large quantities of cultured cells. Moreover, methods such as solvent
228
extraction would destroy the association between EE2 and cf-SWCNTs during extraction.
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The response of MVLN cells is highly sensitive and can indicate the estrogenic activity of
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EE2 at pg/L levels. In order to measure the EE2 desorption during exposure, a linear 12
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correlation (r2 = 0.997) in the range of EE2 concentration from 2.96 × 10-2 to 2.96 ng/L was
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obtained (Figure 3B). Therefore, within this range, the EE2 concentration that induced
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expression of luciferase, named effective concentration, can be calculated. So we calculated
234
the effective concentrations of adsorbed EE2 during exposure. For example, the final
235
exposure concentration of EE2 for CSE-1 was 592 ng/L, but its effective concentration of
236
EE2 was calculated as 0.89 ng/L, indicating that only 0.15% of the adsorbed EE2 was
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desorbed from cf-SWCNTs. As shown in Figure 3C, the desorption rates of all test groups
238
(estimated from the effective concentration and adsorption concentration) were less than
239
0.2%, indicating most EE2 remained on cf-SWCNTs during exposure.
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It has been reported that fullerene (C60) co-exposed with benzo[α]pyrene (Bap) increases
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Bap accumulation in cells.30 The uptake of cf-SWCNTs during phagocytosis, diffusion and
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endocytosis31 may facilitate the transport of EE2 through cell membranes, then influenced the
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bioactivity of EE2. However, little is known whether EE2 could be adsorbed onto
244
cf-SWCNTs in cell culture medium during co-exposure, and then influenced its bioactivity.
245
Here cf-SWCNTs were added to the cell culture medium containing EE2, then the estrogenic
246
activity of EE2 was measured after 48 h exposure. There were no significant changes in the
247
relative estrogenic activity between all cf-SWCNTs+EE2 groups and EE2 control (29.6 ng/L)
248
(p > 0.05) (Figure S2A), indicating the bioactivity of EE2 was not inhibited by additional
249
cf-SWCNTs. We also measured the estrogenic activities of cf-SWCNTs+EE2 groups and
250
EE2 control every 12 h during exposure, and showed a similar tendency towards variation in
251
estrogenic activity (Figure S2B), indicating that the response speed of EE2 were not
252
influenced by additional cf-SWCNTs during co-exposure. Because it is known that a number 13
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of proteins (e.g., serum albumin, immunoglobulin, streptavidin) are capable of nonspecific
254
binding to carbon nanotube surface,32 that EE2 could not adsorbed onto cf-SWCNTs during
255
co-exposure may be due to the competitive adsorption of proteins in cell culture medium.
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Competitive adsorption of PAHs with other organic chemicals such as dissolved organic
257
matter and surfactants has previously been studied.33, 34 Xing et al. reported that the release of
258
residual HOC from CNTs could be enhanced by biomolecules such as pepsin and bile salts in
259
the digestive tract, thus increasing the bioaccessibility of adsorbed phenanthrene and possibly
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the overall toxicity of phenanthrene associated with CNTs.24 It is unknown whether the
261
biomolecules in cell culture or cytoplasm could lead to the desorption of EE2 from
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cf-SWCNTs. Here we investigated the bioactivity change of adsorbed EE2 in culture medium
263
containing different concentrations of FBS under extended incubation time. As shown in
264
Figure 4, there were no obvious changes in the estrogenic activity of adsorbed EE2 in culture
265
medium containing 5-20% FBS over 10 day’s incubation. It suggests that the adsorbed EE2
266
of cf-SWCNTs is highly stable in cell culture medium. However, the bioactivity of adsorbed
267
EE2 on cf-SWCNTs may be prolonged and cause chronic situation in biological or
268
environmental relevant setting because of its high stability and possible desorption.
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Desorption of toxic chemicals from CNTs is very important for evaluating the
270
environmental and health risk of chemicals, because desorption renders these chemicals
271
mobile and bioavailable in the environment or in organisms. Previous study has shown
272
reversible adsorption of PAHs on CNTs.23 However, irreversible hysteresis has been
273
observed for some organic chemicals such as bisphenol A and EE2 upon their desorption
274
from CNTs into water.5 It has been proposed that the irreversible hysteresis of EE2 from 14
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CNTs was attributed to the rearrangement of bundles or aggregates of CNTs.5, 35 However, a
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few adsorbed EE2 (
