Subscriber access provided by Lulea University of Technology
Article
Effect of ingested tungsten oxide (WOX) nanofibers on digestive gland tissue of Porcellio scaber (Isopoda, Crustacea): Fourier transform infrared (FTIR) imaging Sara Novak, Damjana Drobne, Lisa Vaccari, Maya Kiskinova, Paolo Ferraris, Giovanni Birarda, Maja Remskar, and Matej Ho#evar Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es402364w • Publication Date (Web): 16 Aug 2013 Downloaded from http://pubs.acs.org on August 28, 2013
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 30
Environmental Science & Technology
Effect of ingested tungsten oxide (WOX) nanofibers on digestive gland tissue of Porcellio scaber (Isopoda, Crustacea): Fourier transform infrared (FTIR) imaging
Sara Novak‡, Damjana Drobne‡,§,†,*, Lisa Vaccari¶, Maya Kiskinova¶, Paolo Ferraris¶, Giovanni Birarda‖, Maja Remškar¥, Matej Hočevar‖
‡
§
Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, Centre of Excellence, Advanced Materials and Technologies for the Future (CO
NAMASTE), Ljubljana, Slovenia, †
Centre of Excellence, Nanoscience and Nanotechnology (Nanocentre), Ljubljana, Slovenia,
¶
Elettra-Sincrotrone Trieste, AREA Science Park, Basovizza, Trieste, Italy,
‖
Lawrence Berkeley National Laboratory, 1 Cyclotron Rd. Berkeley CA, USA,
¥
Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000
Ljubljana, Slovenia, ‖
Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia
* Corresponding author:
[email protected] 1 ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 30
1
Abstract
2
Tungsten nanofibers are recognised as biologically potent. We study deviations in molecular
3
composition between normal and digestive gland tissue of WOx nanofibers (nano-WOx) fed
4
invertebrate Porcellio scaber (Iosopda, Crustacea) and revealed mechanisms of nano-WOx
5
effect in vivo. Fourier transform infrared (FTIR) imaging performed on digestive gland
6
epithelium was supplemented by toxicity and cytotoxicity analyses as well as scanning
7
electron microscopy (SEM) of the surface of digestive gland epithelium. The difference in the
8
spectra of the WOx-treated and control cells showed up in the central region of the cells and
9
were related to a changed protein to lipid ratio, lipid peroxidation and structural changes of
10
nucleic acids. The conventional toxicity parameters failed to show toxic effects of nano-WOx,
11
whereas the cytotoxicity biomarkers and SEM investigation of digestive gland epithelium
12
indicated sporadic effects of nanofibers. Since toxicological and cytological measurements
13
did not highlight severe effects, the biochemical alterations evidenced by FTIR imaging have
14
been explained as the result of cell protection (acclimation) mechanisms to unfavourable
15
conditions and indication of non-homeostatic state, which can lead to toxic effects.
16 17 18 19 20
2 ACS Paragon Plus Environment
Page 3 of 30
21
Environmental Science & Technology
Introduction
22
The amount of tungsten based products is increasing substantially.1 Tungsten oxides
23
(WO3, WO2, and WOx), which have attractive semiconductor properties, have been
24
considered for many important applications including optical devices, gas sensors,
25
electrochromic windows, and photocatalysts.2 Anthropogenic activities significantly increase
26
tungsten concentration in environmental systems.3 Tungsten powder mixed with soils at rates
27
higher than 1% on a mass basis, trigger changes in soil microbial communities resulting in the
28
death of a substantial portion of the bacterial component and an increase of the fungal
29
biomass. It also induces the death of red worms and plants.3
30
Synthesis of tungsten oxides can be also accompanied by release of fiber-like
31
nanoparticles which raises safety concerns reminiscent of those associated with asbestos
32
fibers, which were found to be highly toxic inducing irreversible health problems.4 WOx
33
nanofibers, whiskers or needles are recognised as being more biologically potent than non-
34
fibrous WOx due to their ability to produce free radical damage in vitro.5 Tungsten carbide
35
particles (WCs) that can cause pneumoconiosisare also well known.6
36
Detection of biological effects can be gained from comparisons of healthy and
37
abnormal tissue, which can be carried out by a variety of physical, biological and biochemical
38
methods. The selection of methods is based on the expected alteration but, when it is
39
necessary to gain a better understanding on molecular and functional changes, methods which
40
can monitor a broad range of structural or functional alterations are required. Among these,
41
Fourier Transform InfraRed (FTIR) microscopy, which uses IR radiation to detect deviations
42
in molecular composition between normal and abnormal tissue is very promising.7 This
43
technique is based on absorption of infrared light that excites molecular vibrations, the
44
intensities of which provide quantitative information, while frequencies give qualitative 3 ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 30
45
knowledge about the nature of these bonds, their structure, and their molecular environment.
46
FTIR microspectroscopy is a label-free, non-destructive and objective tool for discriminating
47
between normal tissues and any alteration. In complex systems such as cells, the main spectral
48
features arise from the backbone vibrations of proteins, lipids, and nucleic acids. The infrared
49
spectrum of cells reflects all these contributions and provides information on the
50
concentration, organization and structure of the most fundamental macromolecules.8
51
Interactions between cells and nanoparticles lead to alteration in cell metabolism,
52
activation of mechanisms that protect against oxidative stress, toxic response and finally cell
53
death.9,
54
oxidation, 11-13 changes in cell membrane fluidity,11 alterations of proteins12, 13 and of DNA.7
55
However, the assessment of nanomaterial toxicity is not always straightforward. The unique
56
chemo-physical properties of nanostructures compared with their bulk counterpart often tune
57
the system response to conventional bio-assays in unpredictable ways, raising many concerns
58
on the outcomes reliability.14 Since the vibrational features of the sample are the diagnostic
59
biomarkers in FTIR spectroscopy and microscopy, the technique is extremely promising in
60
this respect and it was indeed successfully exploited for highlighting the biomolecular
61
mechanisms of multiwalled carbon nanotube toxicity on both prokaryotic and eukaryotic cells
62
by combining synchrotron radiation FTIR imaging and multivariate analysis.15 Some authors
63
also demonstrated the technique sensitivity to cellular changes induced by environmentally-
64
relevant concentrations of contaminants.15, 16 Overall, FTIR spectroscopic approaches allows
65
studying effects of different substances and stress agents in non-invasively, non-destructively
66
and in a high-throughput fashion. 15-17
10
Many papers report effects of nano and microparticles on lipid and protein
67
In our study the capabilities of FTIR imaging has been complemented with
68
conventional bioassays to study deviations in molecular composition between normal
4 ACS Paragon Plus Environment
Page 5 of 30
Environmental Science & Technology
69
digestive gland tissue and digestive gland tissue of WOx nanofibers (nano-WOx) fed animals.
70
The aim this work was to reveal the mechanism of in vivo effect of nano-WOx on digestive
71
gland cells of a model organism, terrestrial invertebrate Porcellio scaber (Isopoda,
72
Crustacea). The advantage of using this organism relies on the possibility to establish a direct
73
correlation between the actual exposure dose to nanofibers and the observed effects at
74
different levels of biological organization, since it is possible to estimate the exposure dose of
75
each test organisms. The feeding parameters are an integrated organism-level response,
76
appropriate evidence of the effects of different chemicals at organism level.18,
77
cellular and biochemical analyses indicate cell level evidence after exposure to chemicals or
78
nanoparticles and to a certain degree their mode of action. Digestive gland cells
79
(hepotopancreas) of terrestrial isopods which combine the functions of pancreas and liver in
80
vertebrates are preferred tissue to study effects of substances with unknown and untargeted
81
action in digestive system.
19
In addition,
82
83
Experimental
84
Model organisms
85
Terrestrial isopods (Porcellio scaber, Isopoda, Crustacea) were collected during July
86
2010 at an uncontaminated location near Ljubljana, Slovenia. The animals were kept in a
87
terrarium filled with a layer of moistened soil and a thick layer of partly decomposed hazelnut
88
tree leaves (Corylus avellana), at a temperature of 20 ± 2 °C and a 16:8-h light:dark
89
photoperiod. Only adult animals of both sexes and weighing more than 30 mg were used in
90
the experiments. If moulting or the presence of marsupia were observed, the animals were not
91
included in the experiment in order to keep the investigated population physiologically as
92
homogenous as possible. 5 ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 30
93
The digestive system of the terrestrial isopod P. scaber is composed of a stomach, four
94
blind-ending digestive gland tubes (hepatopancreas) and a gut. Food enters the digestive
95
glands directly via a short stomach or after the reflux from the gut and ingested material is
96
mixed with digestive fluids.
97
Synthesis and characterization of WOx nanofibers
98
The WOx nanofibers were synthesized by a chemical transport reaction20 at Jozef
99
Stefan Institute, Condensed Matter Physics Department, from tungsten powder (99.9%,
100
Sigma-Aldrich) and WO3 powder (99.9%, Sigma-Aldrich) in the stoichiometric ratio of
101
WO2,86. Iodine (99.8%) in a volume fraction of 3.2 mg/cm3 was added as a transport agent.
102
The material was transported from the source (hot zone at 1123 K) to the growth zone (1009
103
K), with a 5.7 K/cm temperature gradient. The produced nanofibers were studied using
104
electron transmission microscopy 200 keV Jeol 2010F, scanning electron microscopy (FE-
105
SEM, Supra 35 VP, Carl Zeiss). XRD spectra were recorded with an AXS D4 Endeavor
106
diffractometer (Bruker Corporation, Karlsruhe,Germany), with Cu Ka1 radiation and a SOL-
107
X energy dispersive detector with the angular range of 2θ from 5° to 75°, a step size of 0.04°
108
and a collection time of 3 to 4 s. Specific surface area of the sample was measured by single
109
point nitrogen absorption technique using a Micromeritics Gemini 2370 instrument.
110
Food preparation
111
In this study the animals consumed particles applied in a suspension to the leaf
112
surface. Hazelnut leaves were collected in an uncontaminated area and dried at room
113
temperature. Dried leaves were cut into pieces of approximately 100 mg. The WOx nanofibers
114
were suspended in distilled water before each experiment to obtain the final concentration of
115
5000 µg nano-WOx/ml. To diminish agglomeration of nanofibers in distilled H2O the
116
suspension was sonicated in an ultrasonic bath for 1h and mixed using a vortex mixer before 6 ACS Paragon Plus Environment
Page 7 of 30
Environmental Science & Technology
117
brushing it on the leaves. In the control group, the leaves were treated only with distilled
118
water. The suspension of nanofibers or the distilled water was brushed onto the abaxial leaf
119
surface to give final nominal concentrations of nanoparticles on the leaves of 5000 µg nano-
120
WOx per gram (dry wt) of leaf and left until dry.
121
Experimental setup
122
Two experiments were performed. The number of animals per group (control or nano-
123
WOx exposed) was 5 in first experiment while 20 in the second one. The set up for both
124
experiments was same. Each individual animal was placed in a 9 cm Petri dish. A single
125
hazelnut leaf segment treated with distilled water or nano-WOx suspension (5000 µg/g of
126
WOx) and placed in each Petri dish was the animal's only food source. Humidity in the Petri
127
dish was maintained by spraying tap water on the internal side of the lid every day. All Petri
128
dishes were kept in a large glass container under controlled conditions in terms of air
129
humidity (≥80%), temperature (21±1°C) and light regime (16:8h light: dark photoperiod).
130
After seven days of exposure to nanofibers, the animals were anaesthetized at low
131
temperature and then decapitated and their digestive glands isolated and subsequently used for
132
different analyses.
133
Feeding parameters were observed in both experiments. Tissue from model organisms
134
from first experiment was used for scanning electron microscopy while digestive gland cell
135
membrane stability assay and FTIR experiments were carried out on the second group.
136
Feeding parameters, weight change and survival
137
After 7 days of exposure of the animals to treated leaves, the faecal pellets and leaves were
138
removed from the Petri dishes, dried at room temperature for 24 h and weighed separately.
139
The feeding rate of isopods was calculated as the mass of leaves consumed per animal’s wet
140
weight per day. The food assimilation efficiency was calculated as the difference between the 7 ACS Paragon Plus Environment
Environmental Science & Technology
Page 8 of 30
141
mass of consumed leaves and mass of faecal pellets divided by the mass of consumed leaves.
142
The weight change of an animal was the difference in its mass from the beginning to the end
143
of the experiment.
144
Digestive gland cell membrane stability assay
145
The cell membrane stability was tested with the modified method previously described
146
by Valant et al.21 A single isolated hepatopancreatic tube was incubated for 5 min in a mixture
147
of the fluorescent dyes acridine orange (AO, Merck) and ethidium bromide (EB, Merck) and
148
then put on a microscope slide. Fresh samples were examined by an Axioimager.Z1
149
fluorescent microscope (Zeiss) and photographed with two different sets of filters. The
150
excitation filter 450 to 490 nm and the emission filter 515 nm (filter set 09) were used to
151
visualize AO and EB stained nuclei, while the excitation filter 365 nm and the emission filter
152
397 nm (filter set 01) were used to visualize nuclei stained with EB only. The cell membrane
153
integrity was assessed by examination of the micrographs. Photographs of intact digestive
154
glands were examined by the same observer twice at intervals of at least 24h. The integrity of
155
cell membrane was assessed visually and classified on the basis of a predefined scale from 0
156
to 9. From preliminary experiments, it was concluded that the non-treated (control) animals
157
show
